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Otter shrew mitogenomes (Afrotheria, Potamogalidae) reconstructed from historical museum skins
(2022)
African otter shrews (Potamogalidae) are Afrotherian mammals adapted to a semi-aquatic lifestyle. Given their rareness, genetic data on otter shrews are limited. By applying laboratory methods tuned for the recovery of archival DNA and an iterative mapping approach, we reconstructed whole mitochondrial genomes of the Giant (Potamogale velox) and Ruwenzori pygmy otter shrew (Micropotamogale ruwenzorii) from historical museum skins. Phylogenetic analyses are consistent with previous reports in recovering a sister relationship between African otter shrews and Malagasy tenrecs. The long branches separating both lineages, however, support their recognition as separate families.
Paleogenomes reveal a complex evolutionary history of late Pleistocene bison in Northeastern China
(2022)
Steppe bison are a typical representative of the Mid-Late Pleistocene steppes of the northern hemisphere.
Despite the abundance of fossil remains, many questions related to their genetic diversity, population structure and dispersal route are still elusive.
Here, we present both near-complete and partial mitochondrial genomes, as well as a partial nuclear genome from fossil bison samples excavated from Late Pleistocene strata in northeastern China.
Maximum-likelihood and Bayesian trees both suggest the bison clade are divided into three maternal haplogroups (A, B and C), and Chinese individuals fall in two of them. Bayesian analysis shows that the split between haplogroup C and the ancestor of haplogroups A and B dates at 326 ky BP (95% HPD: 397-264 ky BP).
In addition, our nuclear phylogenomic tree also supports a basal position for the individual carrying haplogroup C. Admixture analyses suggest that CADG467 (haplogroup C) has a similar genetic structure to steppe bison from Siberia (haplogroup B).
Our new findings indicate that the genetic diversity of Pleistocene bison was probably even higher than previously thought and that northeastern Chinese populations of several mammalian species, including Pleistocene bison, were genetically distinct.
The subgenus Laurentomantis in the genus Gephyromantis contains some of the least known amphibian species of Madagascar. The six currently valid nominal species are rainforest frogs known from few individuals, hampering a full understanding of the species diversity of the clade. We assembled data on specimens collected during field surveys over the past 30 years and integrated analysis of mitochondrial and nuclear-encoded genes of 88 individuals, a comprehensive bioacoustic analysis, and morphological comparisons to delimit a minimum of nine species-level lineages in the subgenus. To clarify the identity of the species Gephyromantis malagasius, we applied a target-enrichment approach to a sample of the 110 year old holotype of Microphryne malagasia Methuen and Hewitt, 1913 to assign this specimen to a lineage based on a mitochondrial DNA barcode. The holotype clustered unambiguously with specimens previously named G. ventrimaculatus. Consequently we propose to consider Trachymantis malagasia ventrimaculatus Angel, 1935 as a junior synonym of Gephyromantis malagasius. Due to this redefinition of G. malagasius, no scientific name is available for any of the four deep lineages of frogs previously subsumed under this name, all characterized by red color ventrally on the hindlimbs. These are here formally named as Gephyromantis fiharimpe sp. nov., G. matsilo sp. nov., G. oelkrugi sp. nov., and G. portonae sp. nov. The new species are distinguishable from each other by genetic divergences of >4% uncorrected pairwise distance in a fragment of the 16S rRNA marker and a combination of morphological and bioacoustic characters. Gephyromantis fiharimpe and G. matsilo occur, respectively, at mid-elevations and lower elevations along a wide stretch of Madagascar's eastern rainforest band, while G. oelkrugi and G. portonae appear to be more range-restricted in parts of Madagascar's North East and Northern Central East regions. Open taxonomic questions surround G. horridus, to which we here assign specimens from Montagne d'Ambre and the type locality Nosy Be; and G. ranjomavo, which contains genetically divergent populations from Marojejy, Tsaratanana, and Ampotsidy.
Etmopteridae (lantern sharks) is the most species-rich family of sharks, comprising more than 50 species.
Many species are described from few individuals, and re-collection of specimens is often hindered by the remoteness of their sampling sites.
For taxonomic studies, comparative morphological analysis of type specimens housed in natural history collections has been the main source of evidence.
In contrast, DNA sequence information has rarely been used.
Most lantern shark collection specimens, including the types, were formalin fixed before long-term storage in ethanol solutions.
The DNA damage caused by both fixation and preservation of specimens has excluded these specimens from DNA sequence-based phylogenetic analyses so far.
However, recent advances in the field of ancient DNA have allowed recovery of wet-collection specimen DNA sequence data.
Here we analyse archival mitochondrial DNA sequences, obtained using ancient DNA approaches, of two wet-collection lantern shark paratype specimens, namely Etmopterus litvinovi and E. pycnolepis, for which the type series represent the only known individuals.
Target capture of mitochondrial markers from single-stranded DNA libraries allows for phylogenetic placement of both species.
Our results suggest synonymy of E. benchleyi with E. litvinovi but support the species status of E. pycnolepis. This revised taxonomy is helpful for future conservation and management efforts, as our results indicate a larger distribution range of E. litvinovi. This study further demonstrates the importance of wet-collection type specimens as genetic resource for taxonomic research.
After initial detection of target archival DNA of a 116-year-old syntype specimen of the smooth lantern shark, Etmopterus pusillus, in a single-stranded DNA library, we shotgun-sequenced additional 9 million reads from this same DNA library. Sequencing reads were used for extracting mitochondrial sequence information for analyses of mitochondrial DNA characteristics and reconstruction of the mitochondrial genome. The archival DNA is highly fragmented. A total of 4599 mitochondrial reads were available for the genome reconstruction using an iterative mapping approach. The resulting genome sequence has 12 times coverage and a length of 16 741 bp. All 37 vertebrate mitochondrial loci plus the control region were identified and annotated. The mitochondrial NADH2 gene was subsequently used to place the syntype haplotype in a network comprising multiple E. pusillus samples from various distant localities as well as sequences from a morphological similar species, the shortfin smooth lantern shark Etmopterus joungi. Results confirm the almost global distribution of E. pusillus and suggest E. joungi to be a junior synonym of E. pusillus. As mitochondrial DNA often represents the only available reference information in non-model organisms, this study illustrates the importance of mitochondrial DNA from an aged, wet collection type specimen for taxonomy.
Insights into the geographical origin and phylogeographical patterns of Paradisaea birds-of-paradise
(2022)
Birds-of-paradise represent a textbook example for geographical speciation and sexual selection. Perhaps the most iconic genus is Paradisaea, which is restricted to New Guinea and a few surrounding islands. Although several species concepts have been applied in the past to disentangle the different entities within this genus, no attempt has been made so far to uncover phylogeographical patterns based on a genetic dataset that includes multiple individuals per species. Here, we applied amplicon sequencing for the mitochondrial fragment Cytb for a total of 69 museum specimens representing all seven Paradisaea species described and inferred both phylogenetic relationships and colonization pathways across the island. Our analyses show that the most recent common ancestor of the diverging lineages within Paradisaea probably originated in the Late Miocene in the eastern part of the Central Range and suggest that tectonic processes played a key role in shaping the diversification and distribution of species. All species were recovered as monophyletic, except for those within the apoda-minor-raggiana clade, which comprises the allopatric and parapatric species P. apoda, P. minor and P. raggiana. The comparatively young divergence times, together with possible instances of mitochondrial introgression and incomplete lineage sorting, suggest recent speciation in this clade.
(1) Background:
Adaptive diversification of complex traits plays a pivotal role in the evolution of organismal diversity. In the freshwater snail genus Tylomelania, adaptive radiations were likely promoted by trophic specialization via diversification of their key foraging organ, the radula.
(2) Methods:
To investigate the molecular basis of radula diversification and its contribution to lineage divergence, we used tissue-specific transcriptomes of two sympatric Tylomelania sarasinorum ecomorphs.
(3) Results:
We show that ecomorphs are genetically divergent lineages with habitat-correlated abundances. Sequence divergence and the proportion of highly differentially expressed genes are significantly higher between radula transcriptomes compared to the mantle and foot. However, the same is not true when all differentially expressed genes or only non-synonymous SNPs are considered. Finally, putative homologs of some candidate genes for radula diversification (hh, arx, gbb) were also found to contribute to trophic specialization in cichlids and Darwin's finches.
(4) Conclusions:
Our results are in line with diversifying selection on the radula driving Tylomelania ecomorph divergence and indicate that some molecular pathways may be especially prone to adaptive diversification, even across phylogenetically distant animal groups.
Ancient genome provides insights into the history of Eurasian lynx in Iberia and Western Europe
(2022)
The Eurasian lynx (Lynx lynx) is one of the most widely distributed felids in the world. However, most of its populations started to decline a few millennia ago. Historical declines have been especially severe in Europe, and particularly in Western Europe, from where the species disappeared in the last few centuries. Here, we analyze the genome of an Eurasian lynx inhabiting the Iberian Peninsula 2500 ya, to gain insights into the phylogeographic position and genetic status of this extinct population. Also, we contextualize previous ancient data in the light of new phylogeographic studies of the species. Our results suggest that the Iberian population is part of an extinct European lineage closely related to the current Carpathian-Baltic lineages. Also, this sample holds the lowest diversity reported for the species so far, and similar to that of the highly endangered Iberian lynx. A combination of historical factors, such as a founder effect while colonizing the peninsula, together with intensified human impacts during the Holocene in the Cantabrian strip, could have led to a genetic impoverishment of the population and precipitated its extinction. Mitogenomic lineages distribution in space and time support the long-term coexistence of several lineages of Eurasian lynx in Western Europe with fluctuating ranges. While mitochondrial sequences related to the lineages currently found in Balkans and Caucasus were predominant during the Pleistocene, those more closely related to the lineage currently distributed in Central Europe prevailed during the Holocene. The use of ancient genomics has proven to be a useful tool to understand the biogeographic pattern of the Eurasian lynx in the past.
Mitochondrial genomes of Late Pleistocene caballine horses from China belong to a separate clade
(2020)
There were several species of Equus in northern China during the Late Pleistocene, including Equus przewalskii and Equus dalianensis. A number of morphological studies have been carried out on E. przewalskii and E. dalianensis, but their evolutionary history is still unresolved. In this study, we retrieved near-complete mitochondrial genomes from E. dalianensis and E. przewalskii specimens excavated from Late Pleistocene strata in northeastern China. Phylogenetic analyses revealed that caballoid horses were divided into two subclades: the New World and the Old World caballine horse subclades. The Old World caballine horses comprise of two deep phylogenetic lineages, with modern and ancient Equus caballus and modern E. przewalskii forming lineage I, and the individuals in this study together with one Yakut specimen forming lineage II. Our results indicate that Chinese Late Pleistocene caballoid horses showed a closer relationship to other Eurasian caballine horses than that to Pleistocene horses from North America. In addition, phylogenetic analyses suggested a close relationship between E. dalianensis and the Chinese fossil E. przewalskii, in agreement with previous researches based on morphological analyses. Interestingly, E. dalianensis and the fossil E. przewalskii were intermixed rather than split into distinct lineages, suggesting either that gene flow existed between these two species or that morphology-based species assignment of palaeontological specimens is not always correct. Moreover, Bayesian analysis showed that the divergence time between the New World and the Old World caballoid horses was at 1.02 Ma (95% CI: 0.86-1.24 Ma), and the two Old World lineages (I & II) split at 0.88 Ma (95% CI: 0.69-1.13 Ma), which indicates that caballoid horses seem to have evolved into different populations in the Old World soon after they migrated from North America via the Bering Land Bridge. Finally, the TMRCA of E. dalianensis was estimated at 0.20 Ma (95% CI: 0.15-0.28 Ma), and it showed a relative low genetic diversity compared with other Equus species.
Consensify
(2020)
A standard practise in palaeogenome analysis is the conversion of mapped short read data into pseudohaploid sequences, frequently by selecting a single high-quality nucleotide at random from the stack of mapped reads. This controls for biases due to differential sequencing coverage, but it does not control for differential rates and types of sequencing error, which are frequently large and variable in datasets obtained from ancient samples. These errors have the potential to distort phylogenetic and population clustering analyses, and to mislead tests of admixture using D statistics. We introduce Consensify, a method for generating pseudohaploid sequences, which controls for biases resulting from differential sequencing coverage while greatly reducing error rates. The error correction is derived directly from the data itself, without the requirement for additional genomic resources or simplifying assumptions such as contemporaneous sampling. For phylogenetic and population clustering analysis, we find that Consensify is less affected by artefacts than methods based on single read sampling. For D statistics, Consensify is more resistant to false positives and appears to be less affected by biases resulting from different laboratory protocols than other frequently used methods. Although Consensify is developed with palaeogenomic data in mind, it is applicable for any low to medium coverage short read datasets. We predict that Consensify will be a useful tool for future studies of palaeogenomes.
Obtaining information about functional details of proteins of extinct species is of critical importance for a better understanding of the real-life appearance, behavior and ecology of these lost entries in the book of life. In this chapter, we discuss the possibilities to retrieve the necessary DNA sequence information from paleogenomic data obtained from fossil specimens, which can then be used to express and subsequently analyze the protein of interest. We discuss the problems specific to ancient DNA, including mis-coding lesions, short read length and incomplete paleogenome assemblies. Finally, we discuss an alternative, but currently rarely used approach, direct PCR amplification, which is especially useful for comparatively short proteins.
Comparing mitogenomic timetrees for two African savannah primate genera (Chlorocebus and Papio)
(2020)
Simultaneous Barcode Sequencing of Diverse Museum Collection Specimens Using a Mixed RNA Bait Set
(2022)
A growing number of publications presenting results from sequencing natural history collection specimens reflect the importance of DNA sequence information from such samples. Ancient DNA extraction and library preparation methods in combination with target gene capture are a way of unlocking archival DNA, including from formalin-fixed wet-collection material. Here we report on an experiment, in which we used an RNA bait set containing baits from a wide taxonomic range of species for DNA hybridisation capture of nuclear and mitochondrial targets for analysing natural history collection specimens. The bait set used consists of 2,492 mitochondrial and 530 nuclear RNA baits and comprises specific barcode loci of diverse animal groups including both invertebrates and vertebrates. The baits allowed to capture DNA sequence information of target barcode loci from 84% of the 37 samples tested, with nuclear markers being captured more frequently and consensus sequences of these being more complete compared to mitochondrial markers. Samples from dry material had a higher rate of success than wet-collection specimens, although target sequence information could be captured from 50% of formalin-fixed samples. Our study illustrates how efforts to obtain barcode sequence information from natural history collection specimens may be combined and are a way of implementing barcoding inventories of scientific collection material.
The bat-eared fox, Otocyon megalotis, is the only member of its genus and is thought to occupy a basal position within the dog family. These factors can lead to challenges in complete mitochondrial reconstructions and accurate phylogenetic positioning. Here, we present the first complete mitochondrial genome of the bat-eared fox recovered using shotgun sequencing and iterative mapping to three distantly related species. Phylogenetic analyses placed the bat-eared fox basal in the Canidae family within the clade including true foxes (Vulpes) and the raccoon dog (Nyctereutes) with high support values. This position is in good agreement with previously published results based on short fragments of mitochondrial and nuclear genes, therefore adding more support to the basal positioning of the bat-eared fox within Canidae.
Ancient DNA of extinct species from the Pleistocene and Holocene has provided valuable evolutionary insights. However, these are largely restricted to mammals and high latitudes because DNA preservation in warm climates is typically poor. In the tropics and subtropics, non-avian reptiles constitute a significant part of the fauna and little is known about the genetics of the many extinct reptiles from tropical islands. We have reconstructed the near-complete mitochondrial genome of an extinct giant tortoise from the Bahamas (Chelonoidis alburyorum) using an approximately 1000-year-old humerus from a water-filled sinkhole (blue hole) on Great Abaco Island. Phylogenetic and molecular clock analyses place this extinct species as closely related to Galapagos (C. niger complex) and Chaco tortoises (C. chilensis), and provide evidence for repeated overseas dispersal in this tortoise group. The ancestors of extant Chelonoidis species arrived in South America from Africa only after the opening of the Atlantic Ocean and dispersed from there to the Caribbean and the Galapagos Islands. Our results also suggest that the anoxic, thermally buffered environment of blue holes may enhance DNA preservation, and thus are opening a window for better understanding evolution and population history of extinct tropical species, which would likely still exist without human impact.
Ancient genomes have revolutionized our understanding of Holocene prehistory and, particularly, the Neolithic transition in western Eurasia. In contrast, East Asia has so far received little attention, despite representing a core region at which the Neolithic transition took place independently ~3 millennia after its onset in the Near East. We report genome-wide data from two hunter-gatherers from Devil’s Gate, an early Neolithic cave site (dated to ~7.7 thousand years ago) located in East Asia, on the border between Russia and Korea. Both of these individuals are genetically most similar to geographically close modern populations from the Amur Basin, all speaking Tungusic languages, and, in particular, to the Ulchi. The similarity to nearby modern populations and the low levels of additional genetic material in the Ulchi imply a high level of genetic continuity in this region during the Holocene, a pattern that markedly contrasts with that reported for Europe.
The performance of hybridization capture combined with next-generation sequencing (NGS) has seen limited investigation with samples from hot and arid regions until now. We applied hybridization capture and shotgun sequencing to recover DNA sequences from bone specimens of ancient-domestic dromedary (Camelus dromedarius) and its extinct ancestor, the wild dromedary from Jordan, Syria, Turkey and the Arabian Peninsula, respectively. Our results show that hybridization capture increased the percentage of mitochondrial DNA (mtDNA) recovery by an average 187-fold and in some cases yielded virtually complete mitochondrial (mt) genomes at multifold coverage in a single capture experiment. Furthermore, we tested the effect of hybridization temperature and time by using a touchdown approach on a limited number of samples. We observed no significant difference in the number of unique dromedary mtDNA reads retrieved with the standard capture compared to the touchdown method. In total, we obtained 14 partial mitochondrial genomes from ancient-domestic dromedaries with 17-95% length coverage and 1.27-47.1-fold read depths for the covered regions. Using whole-genome shotgun sequencing, we successfully recovered endogenous dromedary nuclear DNA (nuDNA) from domestic and wild dromedary specimens with 1-1.06-fold read depths for covered regions. Our results highlight that despite recent methodological advances, obtaining ancient DNA (aDNA) from specimens recovered from hot, arid environments is still problematic. Hybridization protocols require specific optimization, and samples at the limit of DNA preservation need multiple replications of DNA extraction and hybridization capture as has been shown previously for Middle Pleistocene specimens.
Near the end of the Pleistocene epoch, populations of the woolly mammoth (Mammuthus primigenius) were distributed across parts of three continents, from western Europe and northern Asia through Beringia to the Atlantic seaboard of North America. Nonetheless, questions about the connectivity and temporal continuity of mammoth populations and species remain unanswered. We use a combination of targeted enrichment and high-throughput sequencing to assemble and interpret a data set of 143 mammoth mitochondrial genomes, sampled from fossils recovered from across their Holarctic range. Our dataset includes 54 previously unpublished mitochondrial genomes and significantly increases the coverage of the Eurasian range of the species. The resulting global phylogeny confirms that the Late Pleistocene mammoth population comprised three distinct mitochondrial lineages that began to diverge ~1.0–2.0 million years ago (Ma). We also find that mammoth mitochondrial lineages were strongly geographically partitioned throughout the Pleistocene. In combination, our genetic results and the pattern of morphological variation in time and space suggest that male-mediated gene flow, rather than large-scale dispersals, was important in the Pleistocene evolutionary history of mammoths.
Eastern Africa has been a prime target for scientific drilling because it is rich in key paleoanthropological sites as well as in paleolakes, containing valuable paleoclimatic information on evolutionary time scales. The Hominin Sites and Paleolakes Drilling Project (HSPDP) explores these paleolakes with the aim of reconstructing environmental conditions around critical episodes of hominin evolution. Identification of biological taxa based on their sedimentary ancient DNA (sedaDNA) traces can contribute to understand past ecological and climatological conditions of the living environment of our ancestors. However, sedaDNA recovery from tropical environments is challenging because high temperatures, UV irradiation, and desiccation result in highly degraded DNA. Consequently, most of the DNA fragments in tropical sediments are too short for PCR amplification. We analyzed sedaDNA in the upper 70 m of the composite sediment core of the HSPDP drill site at Chew Bahir for eukaryotic remnants. We first tested shotgun high throughput sequencing which leads to metagenomes dominated by bacterial DNA of the deep biosphere, while only a small fraction was derived from eukaryotic, and thus probably ancient, DNA. Subsequently, we performed cross-species hybridization capture of sedaDNA to enrich ancient DNA (aDNA) from eukaryotic remnants for paleoenvironmental analysis, using established barcoding genes (cox1 and rbcL for animals and plants, respectively) from 199 species that may have had relatives in the past biosphere at Chew Bahir. Metagenomes yielded after hybridization capture are richer in reads with similarity to cox1 and rbcL in comparison to metagenomes without prior hybridization capture. Taxonomic assignments of the reads from these hybridization capture metagenomes also yielded larger fractions of the eukaryotic domain. For reads assigned to cox1, inferred wet periods were associated with high inferred relative abundances of putative limnic organisms (gastropods, green algae), while inferred dry periods showed increased relative abundances for insects. These findings indicate that cross-species hybridization capture can be an effective approach to enhance the information content of sedaDNA in order to explore biosphere changes associated with past environmental conditions, enabling such analyses even under tropical conditions.
An ‛Aukward’ tale
(2017)
One hundred and seventy-three years ago, the last two Great Auks, Pinguinus impennis, ever reliably seen were killed. Their internal organs can be found in the collections of the Natural History Museum of Denmark, but the location of their skins has remained a mystery. In 1999, Great Auk expert Errol Fuller proposed a list of five potential candidate skins in museums around the world. Here we take a palaeogenomic approach to test which—if any—of Fuller’s candidate skins likely belong to either of the two birds. Using mitochondrial genomes from the five candidate birds (housed in museums in Bremen, Brussels, Kiel, Los Angeles, and Oldenburg) and the organs of the last two known individuals, we partially solve the mystery that has been on Great Auk scholars’ minds for generations and make new suggestions as to the whereabouts of the still-missing skin from these two birds.
Present-day domestic horses are immensely diverse in their maternally inherited mitochondrial DNA, yet they show very little variation on their paternally inherited Y chromosome. Although it has recently been shown that Y chromosomal diversity in domestic horses was higher at least until the Iron Age, when and why this diversity disappeared remain controversial questions. We genotyped 16 recently discovered Y chromosomal single-nucleotide polymorphisms in 96 ancient Eurasian stallions spanning the early domestication stages (Copper and Bronze Age) to the Middle Ages. Using this Y chromosomal time series, which covers nearly the entire history of horse domestication, we reveal how Y chromosomal diversity changed over time. Our results also show that the lack of multiple stallion lineages in the extant domestic population is caused by neither a founder effect nor random demographic effects but instead is the result of artificial selection-initially during the Iron Age by nomadic people from the Eurasian steppes and later during the Roman period. Moreover, the modern domestic haplotype probably derived from another, already advantageous, haplotype, most likely after the beginning of the domestication. In line with recent findings indicating that the Przewalski and domestic horse lineages remained connected by gene flow after they diverged about 45,000 years ago, we present evidence for Y chromosomal introgression of Przewalski horses into the gene pool of European domestic horses at least until medieval times.
Hyenas (family Hyaenidae), as the sister group to cats (family Felidae), represent a deeply diverging branch within the cat-like carnivores (Feliformia). With an estimated population size of <10,000 individuals worldwide, the brown hyena (Parahyaena brunnea) represents the rarest of the four extant hyena species and has been listed as Near Threatened by the IUCN. Here, we report a high-coverage genome from a captive bred brown hyena and both mitochondrial and low-coverage nuclear genomes of 14 wild-caught brown hyena individuals from across southern Africa. We find that brown hyena harbor extremely low genetic diversity on both the mitochondrial and nuclear level, most likely resulting from a continuous and ongoing decline in effective population size that started similar to 1 Ma and dramatically accelerated towards the end of the Pleistocene. Despite the strikingly low genetic diversity, we find no evidence of inbreeding within the captive bred individual and reveal phylogeographic structure, suggesting the existence of several potential subpopulations within the species.
Taxonomy plays a central role in biological sciences. It provides a communication system for scientists as it aims to enable correct identification of the studied organisms. As a consequence, species descriptions should seek to include as much available information as possible at species level to follow an integrative concept of 'taxonomics'. Here, we describe the cryptic species Epimeria frankei sp. nov. from the North Sea, and also redescribe its sister species, Epimeria cornigera. The morphological information obtained is substantiated by DNA barcodes and complete nuclear 18S rRNA gene sequences. In addition, we provide, for the first time, full mitochondrial genome data as part of a metazoan species description for a holotype, as well as the neotype. This study represents the first successful implementation of the recently proposed concept of taxonomics, using data from high-throughput technologies for integrative taxonomic studies, allowing the highest level of confidence for both biodiversity and ecological research.
The radula is the central foraging organ and apomorphy of the Mollusca. However, in contrast to other innovations, including the mollusk shell, genetic underpinnings of radula formation remain virtually unknown. Here, we present the first radula formative tissue transcriptome using the viviparous freshwater snail Tylomelania sarasinorum and compare it to foot tissue and the shell-building mantle of the same species. We combine differential expression, functional enrichment, and phylostratigraphic analyses to identify both specific and shared genetic underpinnings of the three tissues as well as their dominant functions and evolutionary origins. Gene expression of radula formative tissue is very distinct, but nevertheless more similar to mantle than to foot. Generally, the genetic bases of both radula and shell formation were shaped by novel orchestration of preexisting genes and continuous evolution of novel genes. A significantly increased proportion of radula-specific genes originated since the origin of stem-mollusks, indicating that novel genes were especially important for radula evolution. Genes with radula-specific expression in our study are frequently also expressed during the formation of other lophotrochozoan hard structures, like chaetae (hes1, arx), spicules (gbx), and shells of mollusks (gbx, heph) and brachiopods (heph), suggesting gene co-option for hard structure formation. Finally, a Lophotrochozoa-specific chitin synthase with a myosin motor domain (CS-MD), which is expressed during mollusk and brachiopod shell formation, had radula-specific expression in our study. CS-MD potentially facilitated the construction of complex chitinous structures and points at the potential of molecular novelties to promote the evolution of different morphological innovations.
Domestic cattle were brought to Spain by early settlers and agricultural societies. Due to missing Neolithic sites in the Spanish region of Galicia, very little is known about this process in this region. We sampled 18 cattle subfossils from different ages and different mountain caves in Galicia, of which 11 were subject to sequencing of the mitochondrial genome and phylogenetic analysis, to provide insight into the introduction of cattle to this region. We detected high similarity between samples from different time periods and were able to compare the time frame of the first domesticated cattle in Galicia to data from the connecting region of Cantabria to show a plausible connection between the Neolithization of these two regions. Our data shows a close relationship of the early domesticated cattle of Galicia and modern cow breeds and gives a general insight into cattle phylogeny. We conclude that settlers migrated to this region of Spain from Europe and introduced common European breeds to Galicia.
Although many large mammal species went extinct at the end of the Pleistocene epoch, their DNA may persist due to past episodes of interspecies admixture. However, direct empirical evidence of the persistence of ancient alleles remains scarce. Here, we present multifold coverage genomic data from four Late Pleistocene cave bears (Ursus spelaeus complex) and show that cave bears hybridized with brown bears (Ursus arctos) during the Pleistocene. We develop an approach to assess both the directionality and relative timing of gene flow. We find that segments of cave bear DNA still persist in the genomes of living brown bears, with cave bears contributing 0.9 to 2.4% of the genomes of all brown bears investigated. Our results show that even though extinction is typically considered as absolute, following admixture, fragments of the gene pool of extinct species can survive for tens of thousands of years in the genomes of extant recipient species.
Targeted capture coupled with high-throughput sequencing can be used to gain information about nuclear sequence variation at hundreds to thousands of loci. Divergent reference capture makes use of molecular data of one species to enrich target loci in other (related) species. This is particularly valuable for nonmodel organisms, for which often no a priori knowledge exists regarding these loci. Here, we have used targeted capture to obtain data for 809 nuclear coding DNA sequences (CDS) in a nonmodel organism, the Eurasian lynx Lynx lynx, using baits designed with the help of the published genome of a related model organism (the domestic cat Felis catus). Using this approach, we were able to survey intraspecific variation at hundreds of nuclear loci in L. lynx across the species’ European range. A large set of biallelic candidate SNPs was then evaluated using a high-throughput SNP genotyping platform (Fluidigm), which we then reduced to a final 96 SNP-panel based on assay performance and reliability; validation was carried out with 100 additional Eurasian lynx samples not included in the SNP discovery phase. The 96 SNP-panel developed from CDS performed very successfully in the identification of individuals and in population genetic structure inference (including the assignment of individuals to their source population). In keeping with recent studies, our results show that genic SNPs can be valuable for genetic monitoring of wildlife species.
The prevalence of contaminant microbial DNA in ancient bone samples represents the principal limiting factor for palaeogenomic studies, as it may comprise more than 99% of DNA molecules obtained. Efforts to exclude or reduce this contaminant fraction have been numerous but also variable in their success. Here, we present a simple but highly effective method to increase the relative proportion of endogenous molecules obtained from ancient bones. Using computed tomography (CT) scanning, we identify the densest region of a bone as optimal for sampling. This approach accurately identifies the densest internal regions of petrous bones, which are known to be a source of high-purity ancient DNA. For ancient long bones, CT scans reveal a high-density outermost layer, which has been routinely removed and discarded prior to DNA extraction. For almost all long bones investigated, we find that targeted sampling of this outermost layer provides an increase in endogenous DNA content over that obtained from softer, trabecular bone. This targeted sampling can produce as much as 50-fold increase in the proportion of endogenous DNA, providing a directly proportional reduction in sequencing costs for shotgun sequencing experiments. The observed increases in endogenous DNA proportion are not associated with any reduction in absolute endogenous molecule recovery. Although sampling the outermost layer can result in higher levels of human contamination, some bones were found to have more contamination associated with the internal bone structures. Our method is highly consistent, reproducible and applicable across a wide range of bone types, ages and species. We predict that this discovery will greatly extend the potential to study ancient populations and species in the genomics era.
Being at the western fringe of Europe, Iberia had a peculiar prehistory and a complex pattern of Neolithization. A few studies, all based on modern populations, reported the presence of DNA of likely African origin in this region, generally concluding it was the result of recent gene flow, probably during the Islamic period. Here, we provide evidence of much older gene flow from Africa to Iberia by sequencing whole genomes from four human remains from northern Portugal and southern Spain dated around 4000 years BP (from the Middle Neolithic to the Bronze Age). We found one of them to carry an unequivocal sub-Saharan mitogenome of most probably West or West-Central African origin, to our knowledge never reported before in prehistoric remains outside Africa. Our analyses of ancient nuclear genomes show small but significant levels of sub-Saharan African affinity in several ancient Iberian samples, which indicates that what we detected was not an occasional individual phenomenon, but an admixture event recognizable at the population level. We interpret this result as evidence of an early migration process from Africa into the Iberian Peninsula through a western route, possibly across the Strait of Gibraltar.
Objective
Plant carnivory is distributed across the tree of life and has evolved at least six times independently, but sequenced and annotated nuclear genomes of carnivorous plants are currently lacking. We have sequenced and structurally annotated the nuclear genome of the carnivorous Roridula gorgonias and that of a non-carnivorous relative, Madeira’s lily-of-the-valley-tree, Clethra arborea, both within the Ericales. This data adds an important resource to study the evolutionary genetics of plant carnivory across angiosperm lineages and also for functional and systematic aspects of plants within the Ericales.
Results
Our assemblies have total lengths of 284 Mbp (R. gorgonias) and 511 Mbp (C. arborea) and show high BUSCO scores of 84.2% and 89.5%, respectively. We used their predicted genes together with publicly available data from other Ericales’ genomes and transcriptomes to assemble a phylogenomic data set for the inference of a species tree. However, groups of orthologs showed a marked absence of species represented by a transcriptome. We discuss possible reasons and caution against combining predicted genes from genome- and transriptome-based assemblies.
Comparing mitogenomic timetrees for two African savannah primate genera (Chlorocebus and Papio)
(2017)
Complete mitochondrial (mtDNA) genomes have proved to be useful in reconstructing primate phylogenies with higher resolution and confidence compared to reconstructions based on partial mtDNA sequences. Here, we analyse complete mtDNA genomes of African green monkeys (genus Chlorocebus), a widely distributed primate genus in Africa representing an interesting phylogeographical model for the evolution of savannah species. Previous studies on partial mtDNA sequences revealed nine major clades, suggesting several cases of para- and polyphyly among Chlorocebus species. However, in these studies, phylogenetic relationships among several clades were not resolved, and divergence times were not estimated. We analysed complete mtDNA genomes for ten Chlorocebus samples representing major mtDNA clades to find stronger statistical support in the phylogenetic reconstruction than in the previous studies and to estimate divergence times. Our results confirmed para- and polyphyletic relationships of most Chlorocebus species, while the support for the phylogenetic relationships between the mtDNA clades increased compared to the previous studies. Our results indicate an initial west-east division in the northern part of the Chlorocebus range with subsequent divergence into north-eastern and southern clades. This phylogeographic scenario contrasts with that for another widespread African savannah primate genus, the baboons (Papio), for which a dispersal from southern Africa into East and West Africa was suggested.
Molecular identification of late and terminal Pleistocene Equus ovodovi from northeastern China
(2019)
The extant diversity of horses (family Equidae) represents a small fraction of that occurring over their evolutionary history. One such lost lineage is the subgenus Sussemionus, which is thought to have become extinct during the Middle Pleistocene. However, recent molecular studies and morphological analysis have revealed that one of their representatives, E. ovodovi, did exist in Siberia during the Late Pleistocene. Fossil materials of E. ovodovi have thus far only been found in Russia. In this study, we extracted DNA from three equid fossil specimens excavated from northeastern China dated at 12,770-12,596, 29,525-28,887 and 40,201-38,848 cal. yBP, respectively, and retrieved three near-complete mitochondrial genomes from the specimens. Phylogenetic analyses cluster the Chinese haplotypes together with previously published Russian E. ovodovi, strongly supporting the assignment of these samples to this taxon. The molecular identification of E. ovodovi in northeastern China extends the known geographical range of this fossil species by several thousand kilometers to the east. The estimated coalescence time of all E. ovodovi haplotypes is approximately 199 Kya, with the Chinese haplotypes coalescing approximately 130 Kya. With a radiocarbon age of 12,770-12,596 cal. yBP, the youngest sample in this study represents the first E. ovodovi sample dating to the terminal Pleistocene, moving the extinction date of this species forwards considerably compared to previously documented fossils. Overall, comparison of our three mitochondrial genomes with the two published ones suggests a genetic diversity similar to several extant species of the genus Equus.
Historically, the giant panda was widely distributed from northern China to southwestern Asia [1]. As a result of range contraction and fragmentation, extant individuals are currently restricted to fragmented mountain ranges on the eastern margin of the Qinghai-Tibet plateau, where they are distributed among three major population clusters [2]. However, little is known about the genetic consequences of this dramatic range contraction. For example, were regions where giant pandas previously existed occupied by ancestors of present-day populations, or were these regions occupied by genetically distinct populations that are now extinct? If so, is there any contribution of these extinct populations to the genomes of giant pandas living today? To investigate these questions, we sequenced the nuclear genome of an similar to 5,000-year-old giant panda from Jiangdongshan, Teng-chong County in Yunnan Province, China. We find that this individual represents a genetically distinct population that diverged prior to the diversification of modern giant panda populations. We find evidence of differential admixture with this ancient population among modern individuals originating from different populations as well as within the same population. We also find evidence for directional gene flow, which transferred alleles from the ancient population into the modern giant panda lineages. A variable proportion of the genomes of extant individuals is therefore likely derived from the ancient population represented by our sequenced individual. Although extant giant panda populations retain reasonable genetic diversity, our results suggest that this represents only part of the genetic diversity this species harbored prior to its recent range contractions.
Horse domestication revolutionized warfare and accelerated travel, trade, and the geographic expansion of languages. Here, we present the largest DNA time series for a non-human organism to date, including genome-scale data from 149 ancient animals and 129 ancient genomes (>= 1-fold coverage), 87 of which are new. This extensive dataset allows us to assess the modem legacy of past equestrian civilisations. We find that two extinct horse lineages existed during early domestication, one at the far western (Iberia) and the other at the far eastern range (Siberia) of Eurasia. None of these contributed significantly to modern diversity. We show that the influence of Persian-related horse lineages increased following the Islamic conquests in Europe and Asia. Multiple alleles associated with elite-racing, including at the MSTN "speed gene," only rose in popularity within the last millennium. Finally, the development of modem breeding impacted genetic diversity more dramatically than the previous millennia of human management.
As limits on O2 availability during submergence impose severe constraints on aerobic respiration, the oxygen binding globin proteins of marine mammals are expected to have evolved under strong evolutionary pressures during their land-to-sea transition. Here, we address this question for the order Sirenia by retrieving, annotating, and performing detailed selection analyses on the globin repertoire of the extinct Steller’s sea cow (Hydrodamalis gigas), dugong (Dugong dugon), and Florida manatee (Trichechus manatus latirostris) in relation to their closest living terrestrial relatives (elephants and hyraxes). These analyses indicate most loci experienced elevated nucleotide substitution rates during their transition to a fully aquatic lifestyle. While most of these genes evolved under neutrality or strong purifying selection, the rate of nonsynonymous/synonymous replacements increased in two genes (Hbz-T1 and Hba-T1) that encode the α-type chains of hemoglobin (Hb) during each stage of life. Notably, the relaxed evolution of Hba-T1 is temporally coupled with the emergence of a chimeric pseudogene (Hba-T2/Hbq-ps) that contributed to the tandemly linked Hba-T1 of stem sirenians via interparalog gene conversion. Functional tests on recombinant Hb proteins from extant and ancestral sirenians further revealed that the molecular remodeling of Hba-T1 coincided with increased Hb–O2 affinity in early sirenians. Available evidence suggests that this trait evolved to maximize O2 extraction from finite lung stores and suppress tissue O2 offloading, thereby facilitating the low metabolic intensities of extant sirenians. In contrast, the derived reduction in Hb–O2 affinity in (sub)Arctic Steller’s sea cows is consistent with fueling increased thermogenesis by these once colossal marine herbivores.
Domestic Bactrian camel (Camelus bactrianus) used to be one of the most important livestock species in Chinese history, as well as the major transport carrier on the ancient Silk Road. However, archeological studies on Chinese C. bactrianus are still limited, and molecular biology research on this species is mainly focused on modern specimens. In this study, we retrieved the complete mitochondrial genome from a C. bactrianus specimen, which was excavated from northwestern China and dated at 1290-1180 cal. Phylogenetic analyses using 18 mitochondrial genomes indicated that the C. bactrianus clade was divided into two maternal lineages. The majority of samples originating from Iran to Japan and Mongolia belong to subclade A1, while our sample together with two Mongolian individuals formed the much smaller subclade A2. Furthermore, the divergence time of these two maternal lineages was estimated as 165 Kya (95% credibility interval 117-222 Kya), this might indicate that several different evolutionary lineages were incorporated into the domestic gene pool during the initial domestication process. Bayesian skyline plot (BSP) analysis a slow increase in female effective population size of C. bactrianus from 5000 years ago, which to the beginning of domestication of C. bactrianus. The present study also revealed that there were extensive exchanges of genetic information among C. bactrianus populations in regions along the Silk Road.
The great auk was once abundant and distributed across the North Atlantic. It is now extinct, having been heavily exploited for its eggs, meat, and feathers. We investigated the impact of human hunting on its demise by integrating genetic data, GPS-based ocean current data, and analyses of population viability. We sequenced complete mitochondrial genomes of 41 individuals from across the species' geographic range and reconstructed population structure and population dynamics throughout the Holocene. Taken together, our data do not provide any evidence that great auks were at risk of extinction prior to the onset of intensive human hunting in the early 16th century. In addition, our population viability analyses reveal that even if the great auk had not been under threat by environmental change, human hunting alone could have been sufficient to cause its extinction. Our results emphasise the vulnerability of even abundant and widespread species to intense and localised exploitation.
Utilising a reconstructed ancestral mitochondrial genome of a clade to design hybridisation capture baits can provide the opportunity for recovering mitochondrial sequences from all its descendent and even sister lineages. This approach is useful for taxa with no extant close relatives, as is often the case for rare or extinct species, and is a viable approach for the analysis of historical museum specimens. Asiatic linsangs (genus Prionodon) exemplify this situation, being rare Southeast Asian carnivores for which little molecular data is available. Using ancestral capture we recover partial mitochondrial genome sequences for seven banded linsangs (P. linsang) from historical specimens, representing the first intraspecific genetic dataset for this species. We additionally assemble a high quality mitogenome for the banded linsang using shotgun sequencing for time-calibrated phylogenetic analysis. This reveals a deep divergence between the two Asiatic linsang species (P. linsang, P. pardicolor), with an estimated divergence of ~12 million years (Ma). Although our sample size precludes any robust interpretation of the population structure of the banded linsang, we recover two distinct matrilines with an estimated tMRCA of ~1 Ma. Our results can be used as a basis for further investigation of the Asiatic linsangs, and further demonstrate the utility of ancestral capture for studying divergent taxa without close relatives.
The genomic changes underlying both early and late stages of horse domestication remain largely unknown. We examined the genomes of 14 early domestic horses from the Bronze and Iron Ages, dating to between similar to 4.1 and 2.3 thousand years before present. We find early domestication selection patterns supporting the neural crest hypothesis, which provides a unified developmental origin for common domestic traits. Within the past 2.3 thousand years, horses lost genetic diversity and archaic DNA tracts introgressed from a now-extinct lineage. They accumulated deleterious mutations later than expected under the cost-of-domestication hypothesis, probably because of breeding from limited numbers of stallions. We also reveal that Iron Age Scythian steppe nomads implemented breeding strategies involving no detectable inbreeding and selection for coat-color variation and robust forelimbs.
Palaeogenomes of Eurasian straight-tusked elephants challenge the current view of elephant evolution
(2017)
The straight-tusked elephants Palaeoloxodon spp. were widespread across Eurasia during the Pleistocene. Phylogenetic reconstructions using morphological traits have grouped them with Asian elephants (Elephas maximus), and many paleontologists place Palaeoloxodon within Elephas. Here, we report the recovery of full mitochondrial genomes from four and partial nuclear genomes from two P. antiquus fossils. These fossils were collected at two sites in Germany, Neumark-Nord and Weimar-Ehringsdorf, and likely date to interglacial periods similar to 120 and similar to 244 thousand years ago, respectively. Unexpectedly, nuclear and mitochondrial DNA analyses suggest that P. antiquus was a close relative of extant African forest elephants (Loxodonta cyclotis). Species previously referred to Palaeoloxodon are thus most parsimoniously explained as having diverged from the lineage of Loxodonta, indicating that Loxodonta has not been constrained to Africa. Our results demonstrate that the current picture of elephant evolution is in need of substantial revision.
The transition from hunting and gathering to farming involved profound cultural and technological changes. In Western and Central Europe, these changes occurred rapidly and synchronously after the arrival of early farmers of Anatolian origin [1-3], who largely replaced the local Mesolithic hunter-gatherers [1, 4-6]. Further east, in the Baltic region, the transition was gradual, with little or no genetic input from incoming farmers [7]. Here we use ancient DNA to investigate the relationship between hunter-gatherers and farmers in the Lower Danube basin, a geographically intermediate area that is characterized by a rapid Neolithic transition but also by the presence of archaeological evidence that points to cultural exchange, and thus possible admixture, between hunter-gatherers and farmers. We recovered four human paleogenomes (1.13 to 4.13 coverage) from Romania spanning a time transect between 8.8 thousand years ago (kya) and 5.4 kya and supplemented them with two Mesolithic genomes (1.73- and 5.33) from Spain to provide further context on the genetic background of Mesolithic Europe. Our results show major Western hunter-gatherer (WHG) ancestry in a Romanian Eneolithic sample with a minor, but sizeable, contribution from Anatolian farmers, suggesting multiple admixture events between hunter-gatherers and farmers. Dietary stableisotope analysis of this sample suggests a mixed terrestrial/ aquatic diet. Our results provide support for complex interactions among hunter-gatherers and farmers in the Danube basin, demonstrating that in some regions, demic and cultural diffusion were not mutually exclusive, but merely the ends of a continuum for the process of Neolithization.
The unusual mix of morphological traits displayed by extinct South American native ungulates (SANUs) confounded both Charles Darwin, who first discovered them, and Richard Owen, who tried to resolve their relationships. Here we report an almost complete mitochondrial genome for the litoptern Macrauchenia. Our dated phylogenetic tree places Macrauchenia as sister to Perissodactyla, but close to the radiation of major lineages within Laurasiatheria. This position is consistent with a divergence estimate of B66Ma (95% credibility interval, 56.64-77.83 Ma) obtained for the split between Macrauchenia and other Panperissodactyla. Combined with their morphological distinctiveness, this evidence supports the positioning of Litopterna (possibly in company with other SANU groups) as a separate order within Laurasiatheria. We also show that, when using strict criteria, extinct taxa marked by deep divergence times and a lack of close living relatives may still be amenable to palaeogenomic analysis through iterative mapping against more distant relatives.
Inactivation of thermogenic UCP1 as a historical contingency in multiple placental mammal clades
(2017)
It is widely accepted that modern pigs were domesticated independently at least twice, and Chinese native pigs are deemed as direct descendants of the first domesticated pigs in the corresponding domestication centers. By analyzing mitochondrial DNA sequences of an extensive sample set spanning 10,000 years, we find that the earliest pigs from the middle Yellow River region already carried the maternal lineages that are dominant in both younger archaeological populations and modern Chinese pigs. Our data set also supports early Neolithic pig utilization and a long-term in situ origin for northeastern Chinese pigs during 8,000-3,500 BP, suggesting a possibly independent domestication in northeast China. Additionally, we observe a genetic replacement in ancient northeast Chinese pigs since 3,500 BP. The results not only provide increasing evidence for pig origin in the middle Yellow River region but also depict an outline for the process of early pig domestication in northeast China.
There is the tendency to assume that endangered species have been both genetically and demographically healthier in the past, so that any genetic erosion observed today was caused by their recent decline. The Iberian lynx (Lynx pardinus) suffered a dramatic and continuous decline during the 20th century, and now shows extremely low genome- and species-wide genetic diversity among other signs of genomic erosion. We analyze ancient (N = 10), historical (N = 245), and contemporary (N = 172) samples with microsatellite and mitogenome data to reconstruct the species' demography and investigate patterns of genetic variation across space and time. Iberian lynx populations transitioned from low but significantly higher genetic diversity than today and shallow geographical differentiation millennia ago, through a structured metapopulation with varying levels of diversity during the last centuries, to two extremely genetically depauperate and differentiated remnant populations by 2002. The historical subpopulations show varying extents of genetic drift in relation to their recent size and time in isolation, but these do not predict whether the populations persisted or went finally extinct. In conclusion, current genetic patterns were mainly shaped by genetic drift, supporting the current admixture of the two genetic pools and calling for a comprehensive genetic management of the ongoing conservation program. This study illustrates how a retrospective analysis of demographic and genetic patterns of endangered species can shed light onto their evolutionary history and this, in turn, can inform conservation actions.
Saber-toothed cats (Machairodontinae) are among the most widely recognized representatives of the now largely extinct Pleistocene megafauna. However, many aspects of their ecology, evolution, and extinction remain uncertain. Although ancient-DNA studies have led to huge advances in our knowledge of these aspects of many other megafauna species (e.g., mammoths and cave bears), relatively few ancient-DNA studies have focused on saber-toothed cats [1-3], and they have been restricted to short fragments of mitochondrial DNA. Here we investigate the evolutionary history of two lineages of saber-toothed cats (Smilodon and Homotherium) in relation to living carnivores and find that the Machairodontinae form a well-supported clade that is distinct from all living felids. We present partial mitochondrial genomes from one S. populator sample and three Homotherium sp. samples, including the only Late Pleistocene Homotherium sample from Eurasia [4]. We confirm the identification of the unique Late Pleistocene European fossil through ancient-DNA analyses, thus strengthening the evidence that Homotherium occurred in Europe over 200,000 years later than previously believed. This in turn forces a re-evaluation of its demography and extinction dynamics. Within the Machairodontinae, we find a deep divergence between Smilodon and Homotherium (similar to 18 million years) but limited diversity between the American and European Homotherium specimens. The genetic data support the hypothesis that all Late Pleistocene (or post-Villafrancian) Homotherium should be considered a single species, H. latidens, which was previously proposed based on morphological data [5, 6].
Abstract
By combining high-throughput sequencing with target enrichment (‘hybridization capture’), researchers are able to obtain molecular data from genomic regions of interest for projects that are otherwise constrained by sample quality (e.g. degraded and contamination-rich samples) or a lack of a priori sequence information (e.g. studies on nonmodel species). Despite the use of hybridization capture in various fields of research for many years, the impact of enrichment conditions on capture success is not yet thoroughly understood. We evaluated the impact of a key parameter – hybridization temperature – on the capture success of mitochondrial genomes across the carnivoran family Felidae. Capture was carried out for a range of sample types (fresh, archival, ancient) with varying levels of sequence divergence between bait and target (i.e. across a range of species) using pools of individually indexed libraries on Agilent SureSelect™ arrays. Our results suggest that hybridization capture protocols require specific optimization for the sample type that is being investigated. Hybridization temperature affected the proportion of on-target sequences following capture: for degraded samples, we obtained the best results with a hybridization temperature of 65 °C, while a touchdown approach (65 °C down to 50 °C) yielded the best results for fresh samples. Evaluation of capture performance at a regional scale (sliding window approach) revealed no significant improvement in the recovery of DNA fragments with high sequence divergence from the bait at any of the tested hybridization temperatures, suggesting that hybridization temperature may not be the critical parameter for the enrichment of divergent fragments.
Background: Skewed body size distributions and the high relative richness of small-bodied taxa are a fundamental property of a wide range of animal clades. The evolutionary processes responsible for generating these distributions are well described in vertebrate model systems but have yet to be explored in detail for other major terrestrial clades. In this study, we explore the macro-evolutionary patterns of body size variation across families of Hexapoda (insects and their close relatives), using recent advances in phylogenetic understanding, with an aim to investigate the link between size and diversity within this ancient and highly diverse lineage. Results: The maximum, minimum and mean-log body lengths of hexapod families are all approximately log-normally distributed, consistent with previous studies at lower taxonomic levels, and contrasting with skewed distributions typical of vertebrate groups. After taking phylogeny and within-tip variation into account, we find no evidence for a negative relationship between diversification rate and body size, suggesting decoupling of the forces controlling these two traits. Likelihood-based modeling of the log-mean body size identifies distinct processes operating within Holometabola and Diptera compared with other hexapod groups, consistent with accelerating rates of size evolution within these clades, while as a whole, hexapod body size evolution is found to be dominated by neutral processes including significant phylogenetic conservatism. Conclusions: Based on our findings we suggest that the use of models derived from well-studied but atypical clades, such as vertebrates may lead to misleading conclusions when applied to other major terrestrial lineages. Our results indicate that within hexapods, and within the limits of current systematic and phylogenetic knowledge, insect diversification is generally unfettered by size-biased macro-evolutionary processes, and that these processes over large timescales tend to converge on apparently neutral evolutionary processes. We also identify limitations on available data within the clade and modeling approaches for the resolution of trees of higher taxa, the resolution of which may collectively enhance our understanding of this key component of terrestrial ecosystems.