Mitochondrial genome fragmentation is correlated with increased rates of molecular evolution.

While mitochondrial genome content and organization is quite diverse across all Eukaryotes, most bilaterian animal mitochondrial genomes (mitogenomes) exhibit highly conserved gene content and organisation, with genes typically encoded on a single circular chromosome. However, many species of parasitic lice (Insecta: Phthiraptera) are among the notable exceptions, having mitogenomes fragmented into multiple circular chromosomes. To better understand the process of mitogenome fragmentation, we conducted a large-scale genomic study of a major group of lice, Amblycera, with extensive taxon sampling. Analyses of the evolution of mitogenome structure across a phylogenomic tree of 90 samples from 53 genera revealed evidence for multiple independent origins of mitogenome fragmentation, some inferred to have occurred less than five million years ago. We leveraged these many independent origins of fragmentation to compare the rates of DNA substitution and gene rearrangement, specifically contrasting branches with fragmented and non-fragmented mitogenomes. We found that lineages with fragmented mitochondrial genomes had significantly higher rates of mitochondrial sequence evolution. In addition, lineages with fragmented mitochondrial genomes were more likely to have mitogenome gene rearrangements than those with single-chromosome mitochondrial genomes. By combining phylogenomics and mitochondrial genomics we provide a detailed portrait of mitogenome evolution across this group of insects with a remarkably unstable mitogenome structure, identifying processes of molecular evolution that are correlated with mitogenome fragmentation.

Genomic data reveal unexpected relatedness between a brown female Eastern Bluebird and her brood.

Because plumage coloration is frequently involved in sexual selection, for both male and female mate choice, birds with aberrant plumage should have fewer mating opportunities and thus lower reproductive output. Here we report an Eastern Bluebird (Sialia sialis) female with a brown phenotype that raised a brood of four chicks to fledging. The brown female and her mate were only related to their social offspring to the second degree and one of the offspring was a half-sibling. We propose four family tree scenarios and discuss their implications (e.g., extra-pair paternity, conspecific brood parasitism). Regardless of the tree, the brown female was able to find a mate, which may have been facilitated by the bottleneck created by the severe snowstorms in February 2021.

Parce que la coloration du plumage est fréquemment impliquée dans la sélection sexuelle, tant pour le choix du partenaire mâle que femelle, les oiseaux avec un plumage aberrant devraient avoir moins d'opportunités d'accouplement et donc un rendement de reproduction réduit. Nous rapportons ici le cas d'une femelle Merlebleu de l'Est (Sialia sialis) avec un phénotype brun qui a élevé une nichée de quatre poussins jusqu'à l'envol. La femelle brune et son partenaire n'étaient liés à leur progéniture sociale qu'au second degré et l'un des poussins était un demi­frère ou une demi­sœur. Nous proposons quatre scenarios d'arbre généalogique et discutons de leurs implications (par exemple, paternité hors couple, parasitisme conspécifique), mais quel que soit l'arbre, la femelle brune a pu trouver un partenaire, ce qui a peut­être été facilité par le goulot d'étranglement créé par les fortes tempêtes de neige en février 2021.

Prevalence and diversity of parasitic bird lice (Insecta: Psocodea) in northeast Arkansas.

Many groups of parasites lack basic information on biodiversity and host associations, which poses challenges for conservation and understanding the ecological relationships between hosts and their parasites. This gap in knowledge is particularly relevant for parasitic species with obscure lifestyles. Ectoparasitc bird lice (Insecta: Psocodea: Phthiraptera) are a group of parasites that has received a relatively substantial research focus, yet patterns of bird-louse relationships and louse diversity remain understudied in many geographic regions, including in parts of the southeastern United States. In this study, we assessed the diversity, prevalence, abundance, and intensity of lice from live and salvaged birds in northeastern Arkansas. We also focused on the frequency of co-occurrence of lice and symbiotic feather mites. Finally, we used nuclear and mitochondrial genes to assess the phylogenic relationships among the most common genera of lice in our sample. We found a total louse prevalence of 10.57% with the highest prevalence on the Passeriformes families Turdidae, Passerellidae, and Parulidae. We also found the louse genera Myrsidea and Brueelia to be the most prevalent and abundant in our sample. Additionally, we reported several novel associations among well-studied bird species. We also found that louse phylogenic patterns tend to reflect host taxonomy and/or ecology. Overall, our results provide important insight into the biodiversity, community structure, and host interactions of parasitic lice from North American birds.

Novel insights into symbiont population structure: Globe-trotting avian feather mites contradict the specialist-generalist variation hypothesis.

Researchers often examine symbiont host specificity as a species-level pattern, but it can also be key to understanding processes occurring at the population level, which are not as well understood. The specialist-generalist variation hypothesis (SGVH) attempts to explain how host specificity influences population-level processes, stating that single-host symbionts (specialists) exhibit stronger population genetic structure than multi-host symbionts (generalists) because of fewer opportunities for dispersal and more restricted gene flow between populations. However, this hypothesis has not been tested in systems with highly mobile hosts, in which population connectivity may vary temporally and spatially. To address this gap, we tested the SGVH on proctophyllodid feather mites found on migratory warblers (family Parulidae) with contrasting host specificities, Amerodectes protonotaria (a host specialist of Protonotaria citrea) and A. ischyros (a host generalist of 17 parulid species). We used a pooled-sequencing approach and a novel workflow to analyse genetic variants obtained from whole genome data. Both mite species exhibited fairly weak population structure overall, and contrary to predictions of the SGVH, the generalist was more strongly structured than the specialist. These results may suggest that specialists disperse more freely among conspecifics, whereas generalists sort according to geography. Furthermore, our results may reflect an unexpected period for mite transmission - during the nonbreeding season of migratory hosts - as mite population structure more closely reflects the distributions of hosts during the nonbreeding season. Our findings alter our current understanding of feather mite biology and highlight the potential for studies to explore factors driving symbiont diversification at multiple evolutionary scales.

Dispersal-Limited Symbionts Exhibit Unexpectedly Wide Variation in Host Specificity.

A fundamental aspect of symbiotic relationships is host specificity, ranging from extreme specialists associated with only a single host species to generalists associated with many different species. Although symbionts with limited dispersal capabilities are expected to be host specialists, some are able to associate with multiple hosts. Understanding the micro- and macro-evolutionary causes of variations in host specificity is often hindered by sampling biases and the limited power of traditional evolutionary markers. Here, we studied feather mites to address the barriers associated with estimates of host specificity for dispersal-limited symbionts. We sampled feather mites (Proctophyllodidae) from a nearly comprehensive set of North American breeding warblers (Parulidae) to study mite phylogenetic relationships and host-symbiont codiversification. We used pooled-sequencing (Pool-Seq) and short-read Illumina technology to interpret results derived from a traditional barcoding gene (cytochrome c oxidase subunit 1) versus 11 protein-coding mitochondrial genes using concatenated and multispecies coalescent approaches. Despite the statistically significant congruence between mite and host phylogenies, mite-host specificity varies widely, and host switching is common regardless of the genetic marker resolution (i.e., barcode vs. multilocus). However, the multilocus approach was more effective than the single barcode in detecting the presence of a heterogeneous Pool-Seq sample. These results suggest that presumed symbiont dispersal capabilities are not always strong indicators of host specificity or of historical host-symbiont coevolutionary events. A comprehensive sampling at fine phylogenetic scales may help to better elucidate the microevolutionary filters that impact macroevolutionary processes regulating symbioses, particularly for dispersal-limited symbionts. [Codiversification; cophylogenetics; feather mites; host switching; pooled sequencing; species delineation; symbiosis, warblers.].

Draft genome assemblies of the avian louse Brueelia nebulosa and its associates using long-read sequencing from an individual specimen.

Sequencing high molecular weight (HMW) DNA with long-read and linked-read technologies has promoted a major increase in more complete genome sequences for nonmodel organisms. Sequencing approaches that rely on HMW DNA have been limited to larger organisms or pools of multiple individuals, but recent advances have allowed for sequencing from individuals of small-bodied organisms. Here, we use HMW DNA sequencing with PacBio long reads and TELL-Seq linked reads to assemble and annotate the genome from a single individual feather louse (Brueelia nebulosa) from a European Starling (Sturnus vulgaris). We assembled a genome with a relatively high scaffold N50 (637 kb) and with BUSCO scores (96.1%) comparable to louse genomes assembled from pooled individuals. We annotated a number of genes (10,938) similar to the human louse (Pediculus humanus) genome. Additionally, calling phased variants revealed that the Brueelia genome is more heterozygous (∼1%) then expected for a highly obligate and dispersal-limited parasite. We also assembled and annotated the mitochondrial genome and primary endosymbiont (Sodalis) genome from the individual louse, which showed evidence for heteroplasmy in the mitogenome and a reduced genome size in the endosymbiont compared to its free-living relative. Our study is a valuable demonstration of the capability to obtain high-quality genomes from individual small, nonmodel organisms. Applying this approach to other organisms could greatly increase our understanding of the diversity and evolution of individual genomes.

Independent evolution of highly variable, fragmented mitogenomes of parasitic lice.

The mitochondrial genomes (mitogenomes) of bilaterian animals are highly conserved structures that usually consist of a single circular chromosome. However, several species of parasitic lice (Insecta: Phthiraptera) possess fragmented mitogenomes, where the mitochondrial genes are present on separate, circular chromosomes. Nevertheless, the extent, causes, and consequences of this structural variation remain poorly understood. Here, we combined new and existing data to better understand the evolution of mitogenome fragmentation in major groups of parasitic lice. We found strong evidence that fragmented mitogenomes evolved many times within parasitic lice and that the level of fragmentation is highly variable, including examples of heteroplasmic arrangements. We also found a significant association between mitochondrial fragmentation and signatures of relaxed selection. Mitochondrial fragmentation was also associated with changes to a lower AT%, possibly due to differences in mutation biases. Together, our results provide a significant advance in understanding the process of mitogenome fragmentation and provide an important perspective on mitochondrial evolution in eukaryotes.

High levels of inbreeding with spatial and host-associated structure in lice of an endangered freshwater seal.

Host-specialist parasites of endangered large vertebrates are in many cases more endangered than their hosts. In particular, low host population densities and reduced among-host transmission rates are expected to lead to inbreeding within parasite infrapopulations living on single host individuals. Furthermore, spatial population structures of directly-transmitted parasites should be concordant with those of their hosts. Using population genomic approaches, we investigated inbreeding and population structure in a host-specialist seal louse (Echinophthirius horridus) infesting the Saimaa ringed seal (Phoca hispida saimensis), which is endemic to Lake Saimaa in Finland, and is one of the most endangered pinnipeds in the world. We conducted genome resequencing of pairs of lice collected from 18 individual Saimaa ringed seals throughout the Lake Saimaa complex. Our analyses showed high genetic similarity and inbreeding between lice inhabiting the same individual seal host, indicating low among-host transmission rates. Across the lake, genetic differentiation among individual lice was correlated with their geographic distance, and assignment analyses revealed a marked break in the genetic variation of the lice in the middle of the lake, indicating substantial population structure. These findings indicate that movements of Saimaa ringed seals across the main breeding areas of the fragmented Lake Saimaa complex may in fact be more restricted than suggested by previous population-genetic analyses of the seals themselves.

Long-distance dispersal of pigeons and doves generated new ecological opportunities for host-switching and adaptive radiation by their parasites.

Adaptive radiation is an important mechanism of organismal diversification and can be triggered by new ecological opportunities. Although poorly studied in this regard, parasites are an ideal group in which to study adaptive radiations because of their close associations with host species. Both experimental and comparative studies suggest that the ectoparasitic wing lice of pigeons and doves have adaptively radiated, leading to differences in body size and overall coloration. Here, we show that long-distance dispersal by dove hosts was central to parasite diversification because it provided new ecological opportunities for parasites to speciate after host-switching. We further show that among extant parasite lineages host-switching decreased over time, with cospeciation becoming the more dominant mode of parasite speciation. Taken together, our results suggest that host dispersal, followed by host-switching, provided novel ecological opportunities that facilitated adaptive radiation by parasites.

Recovery and analysis of ancient beetle DNA from subfossil packrat middens using high-throughput sequencing.

The study of ancient DNA is revolutionizing our understanding of paleo-ecology and the evolutionary history of species. Insects are essential components in many ecosystems and constitute the most diverse group of animals. Yet they are largely neglected in ancient DNA studies. We report the results of the first targeted investigation of insect ancient DNA to positively identify subfossil insects to species, which includes the recovery of endogenous content from samples as old as ~ 34,355 ybp. Potential inhibitors currently limiting widespread research on insect ancient DNA are discussed, including the lack of closely related genomic reference sequences (decreased mapping efficiency) and the need for more extensive collaborations with insect taxonomists. The advantages of insect-based studies are also highlighted, especially in the context of understanding past climate change. In this regard, insect remains from ancient packrat middens are a rich and largely uninvestigated resource for exploring paleo-ecology and species dynamics over time.

Structure, gene order, and nucleotide composition of mitochondrial genomes in parasitic lice from Amblycera.

Parasitic lice have unique mitochondrial (mt) genomes characterized by rearranged gene orders, variable genome structures, and less AT content compared to most other insects. However, relatively little is known about the mt genomes of Amblycera, the suborder sister to all other parasitic lice. Comparing among nine different genera (including representative of all seven families), we show that Amblycera have variable and highly rearranged mt genomes. Some genera have fragmented genomes that vary considerably in length, whereas others have a single mt chromosome. Notably, these genomes are more AT-biased than most other lice. We also recover genus-level phylogenetic relationships among Amblycera that are consistent with those reported from large nuclear datasets, indicating that mt sequences are reliable for reconstructing evolutionary relationships in Amblycera. However, gene order data cannot reliably recover these same relationships. Overall, our results suggest that the mt genomes of lice, already know to be distinctive, are even more variable than previously thought.

Combining Nuclear and Mitochondrial Loci Provides Phylogenetic Information in the Philopterus Complex of Lice (Psocodea: Ischnocera: Philopteridae).

The Philopterus Complex includes several lineages of lice that occur on birds. The complex includes the genera Philopterus (Nitzsch, 1818; Psocodea: Philopteridae), Philopteroides (Mey, 2004; Psocodea: Philopteridae), and many other lineages that have sometimes been regarded as separate genera. Only a few studies have investigated the phylogeny of this complex, all of which are based on morphological data. Here we evaluate the utility of nuclear and mitochondrial loci for recovering the phylogeny within this group. We obtained phylogenetic trees from 39 samples of the Philopterus Complex (Psocodea: Philopteridae), using sequences of two nuclear (hyp and TMEDE6) and one mitochondrial (COI) marker. We evaluated trees derived from these genes individually as well as from concatenated sequences. All trees show 20 clearly demarcated taxa (i.e., putative species) divided into five well-supported clades. Percent sequence divergence between putative species (~5-30%) for the COI gene tended to be much higher than those for the nuclear genes (~1-15%), as expected. In cases where species are described, the lineages identified based on molecular divergence correspond to morphologically defined species. In some cases, species that are host generalists exhibit additional underlying genetic variation and such cases need to be explored by further future taxonomic revisions of the Philopterus Complex.

Phylogenomics of Parasitic and Nonparasitic Lice (Insecta: Psocodea): Combining Sequence Data and Exploring Compositional Bias Solutions in Next Generation Data Sets.

The insect order Psocodea is a diverse lineage comprising both parasitic (Phthiraptera) and nonparasitic members (Psocoptera). The extreme age and ecological diversity of the group may be associated with major genomic changes, such as base compositional biases expected to affect phylogenetic inference. Divergent morphology between parasitic and nonparasitic members has also obscured the origins of parasitism within the order. We conducted a phylogenomic analysis on the order Psocodea utilizing both transcriptome and genome sequencing to obtain a data set of 2370 orthologous genes. All phylogenomic analyses, including both concatenated and coalescent methods suggest a single origin of parasitism within the order Psocodea, resolving conflicting results from previous studies. This phylogeny allows us to propose a stable ordinal level classification scheme that retains significant taxonomic names present in historical scientific literature and reflects the evolution of the group as a whole. A dating analysis, with internal nodes calibrated by fossil evidence, suggests an origin of parasitism that predates the K-Pg boundary. Nucleotide compositional biases are detected in third and first codon positions and result in the anomalous placement of the Amphientometae as sister to Psocomorpha when all nucleotide sites are analyzed. Likelihood-mapping and quartet sampling methods demonstrate that base compositional biases can also have an effect on quartet-based methods.[Illumina; Phthiraptera; Psocoptera; quartet sampling; recoding methods.].

Comparing rates of introgression in parasitic feather lice with differing dispersal capabilities.

Organisms vary in their dispersal abilities, and these differences can have important biological consequences, such as impacting the likelihood of hybridization events. However, there is still much to learn about the factors influencing hybridization, and specifically how dispersal ability affects the opportunities for hybridization. Here, using the ecological replicate system of dove wing and body lice (Insecta: Phthiraptera), we show that species with higher dispersal abilities exhibited increased genomic signatures of introgression. Specifically, we found a higher proportion of introgressed genomic reads and more reticulated phylogenetic networks in wing lice, the louse group with higher dispersal abilities. Our results are consistent with the hypothesis that differences in dispersal ability might drive the extent of introgression through hybridization.

Lousy grouse: Comparing evolutionary patterns in Alaska galliform lice to understand host evolution and host-parasite interactions.

Understanding both sides of host-parasite relationships can provide more complete insights into host and parasite biology in natural systems. For example, phylogenetic and population genetic comparisons between a group of hosts and their closely associated parasites can reveal patterns of host dispersal, interspecies interactions, and population structure that might not be evident from host data alone. These comparisons are also useful for understanding factors that drive host-parasite coevolutionary patterns (e.g., codivergence or host switching) over different periods of time. However, few studies have compared the evolutionary histories between multiple groups of parasites from the same group of hosts at a regional geographic scale. Here, we used genomic data to compare phylogenomic and population genomic patterns of Alaska ptarmigan and grouse species (Aves: Tetraoninae) and two genera of their associated feather lice: Lagopoecus and Goniodes. We used whole-genome sequencing to obtain hundreds of genes and thousands of single-nucleotide polymorphisms (SNPs) for the lice and double-digest restriction-associated DNA sequences to obtain SNPs from Alaska populations of two species of ptarmigan. We found that both genera of lice have some codivergence with their galliform hosts, but these relationships are primarily characterized by host switching and phylogenetic incongruence. Population structure was also uncorrelated between the hosts and lice. These patterns suggest that grouse, and ptarmigan in particular, share habitats and have likely had historical and ongoing dispersal within Alaska. However, the two genera of lice also have sufficient dissimilarities in the relationships with their hosts to suggest there are other factors, such as differences in louse dispersal ability, that shape the evolutionary patterns with their hosts.

Mitochondrial genomes of Columbicola feather lice are highly fragmented, indicating repeated evolution of minicircle-type genomes in parasitic lice.

Most animals have a conserved mitochondrial genome structure composed of a single chromosome. However, some organisms have their mitochondrial genes separated on several smaller circular or linear chromosomes. Highly fragmented circular chromosomes ("minicircles") are especially prevalent in parasitic lice (Insecta: Phthiraptera), with 16 species known to have between nine and 20 mitochondrial minicircles per genome. All of these species belong to the same clade (mammalian lice), suggesting a single origin of drastic fragmentation. Nevertheless, other work indicates a lesser degree of fragmentation (2-3 chromosomes/genome) is present in some avian feather lice (Ischnocera: Philopteridae). In this study, we tested for minicircles in four species of the feather louse genus Columbicola (Philopteridae). Using whole genome shotgun sequence data, we applied three different bioinformatic approaches for assembling the Columbicola mitochondrial genome. We further confirmed these approaches by assembling the mitochondrial genome of Pediculus humanus from shotgun sequencing reads, a species known to have minicircles. Columbicola spp. genomes are highly fragmented into 15-17 minicircles between ∼1,100 and ∼3,100 bp in length, with 1-4 genes per minicircle. Subsequent annotation of the minicircles indicated that tRNA arrangements of minicircles varied substantially between species. These mitochondrial minicircles for species of Columbicola represent the first feather lice (Philopteridae) for which minicircles have been found in a full mitochondrial genome assembly. Combined with recent phylogenetic studies of parasitic lice, our results provide strong evidence that highly fragmented mitochondrial genomes, which are otherwise rare across the Tree of Life, evolved multiple times within parasitic lice.

Extensive in situ radiation of feather lice on tinamous.

Tinamous host the highest generic diversity of lice of any group of birds, as well as hosting representatives of all four avian feather louse ecomorphs. Although the generic diversity of tinamou feather lice is well documented, few attempts have been made to reconstruct the phylogenetic relationships among these lice. To test whether tinamou feather lice form a monophyletic group as a whole, we used whole-genome sequencing to estimate a higher-level phylogeny of tinamou feather lice, together with a broad diversity of other avian feather louse groups. In total, we analysed sequences from over 1000 genes for 48 genera of avian lice using both concatenated and coalescent approaches to estimate the phylogeny of this diverse group of avian feather lice. Although the body louse ecomorph of tinamou feather lice formed a monophyletic group, they did not strictly form a monophyletic group together with the other three ecomorphs of tinamou feather lice. In particular, a clade comprised of several feather louse genera, mainly from South America, is nested phylogenetically within tinamou lice, which also have their main centre of diversity in South America. These results suggest in situ radiation of these parasites in South America.

Rearrangement and evolution of mitochondrial genomes in Thysanoptera (Insecta).

Prior to this study, complete mitochondrial genomes from Order Thysanoptera were restricted to a single family, the Thripidae, resulting in a biased view of their evolution. Here we present the sequences for the mitochondrial genomes of four additional thrips species, adding three extra families and an additional subfamily, thus greatly improving taxonomic coverage. Thrips mitochondrial genomes are marked by high rates of gene rearrangement, duplications of the control region and tRNA mutations. Derived features of mitochondrial tRNAs in thrips include gene duplications, anticodon mutations, loss of secondary structures and high gene translocation rates. Duplicated control regions are found in the Aeolothripidae and the 'core' Thripinae clade but do not appear to promote gene rearrangement as previously proposed. Phylogenetic analysis of thrips mitochondrial sequence data supports the monophyly of two suborders, a sister-group relationship between Stenurothripidae and Thripidae, and suggests a novel set of relationships between thripid genera. Ancestral state reconstructions indicate that genome rearrangements are common, with just eight gene blocks conserved between any thrips species and the ancestral insect mitochondrial genome. Conversely, 71 derived rearrangements are shared between at least two species, and 24 of these are unambiguous synapomorphies for clades identified by phylogenetic analysis. While the reconstructed sequence of genome rearrangements among the protein-coding and ribosomal RNA genes could be inferred across the phylogeny, direct inference of phylogeny from rearrangement data in MLGO resulted in a highly discordant set of relationships inconsistent with both sequence-based phylogenies and previous morphological analysis. Given the demonstrated rates of genomic evolution within thrips, extensive sampling is needed to fully understand these phenomena across the order.

Extensive host-switching of avian feather lice following the Cretaceous-Paleogene mass extinction event.

Nearly all lineages of birds host parasitic feather lice. Based on recent phylogenomic studies, the three major lineages of modern birds diverged from each other before the Cretaceous-Paleogene (K-Pg) mass extinction event. In contrast, studies of the phylogeny of feather lice on birds, indicate that these parasites diversified largely after this event. However, these studies were unable to reconstruct the ancestral avian host lineage for feather lice. Here we use genome sequences of a broad diversity of lice to reconstruct a phylogeny based on 1,075 genes. By comparing this louse evolutionary tree to the avian host tree, we show that feather lice began diversifying on the common ancestor of waterfowl and landfowl, then radiated onto other avian lineages by extensive host-switching. Dating analyses and cophylogenetic comparisons revealed that two of three lineages of birds that diverged before the K-Pg boundary acquired their feather lice after this event via host-switching.

Fully automated sequence alignment methods are comparable to, and much faster than, traditional methods in large data sets: an example with hepatitis B virus.

Aligning sequences for phylogenetic analysis (multiple sequence alignment; MSA) is an important, but increasingly computationally expensive step with the recent surge in DNA sequence data. Much of this sequence data is publicly available, but can be extremely fragmentary (i.e., a combination of full genomes and genomic fragments), which can compound the computational issues related to MSA. Traditionally, alignments are produced with automated algorithms and then checked and/or corrected "by eye" prior to phylogenetic inference. However, this manual curation is inefficient at the data scales required of modern phylogenetics and results in alignments that are not reproducible. Recently, methods have been developed for fully automating alignments of large data sets, but it is unclear if these methods produce alignments that result in compatible phylogenies when compared to more traditional alignment approaches that combined automated and manual methods. Here we use approximately 33,000 publicly available sequences from the hepatitis B virus (HBV), a globally distributed and rapidly evolving virus, to compare different alignment approaches. Using one data set comprised exclusively of whole genomes and a second that also included sequence fragments, we compared three MSA methods: (1) a purely automated approach using traditional software, (2) an automated approach including by eye manual editing, and (3) more recent fully automated approaches. To understand how these methods affect phylogenetic results, we compared resulting tree topologies based on these different alignment methods using multiple metrics. We further determined if the monophyly of existing HBV genotypes was supported in phylogenies estimated from each alignment type and under different statistical support thresholds. Traditional and fully automated alignments produced similar HBV phylogenies. Although there was variability between branch support thresholds, allowing lower support thresholds tended to result in more differences among trees. Therefore, differences between the trees could be best explained by phylogenetic uncertainty unrelated to the MSA method used. Nevertheless, automated alignment approaches did not require human intervention and were therefore considerably less time-intensive than traditional approaches. Because of this, we conclude that fully automated algorithms for MSA are fully compatible with older methods even in extremely difficult to align data sets. Additionally, we found that most HBV diagnostic genotypes did not correspond to evolutionarily-sound groups, regardless of alignment type and support threshold. This suggests there may be errors in genotype classification in the database or that HBV genotypes may need a revision.