Specimen collection is essential for modern science.

Natural history museums are vital repositories of specimens, samples and data that inform about the natural world; this Formal Comment revisits a Perspective that advocated for the adoption of compassionate collection practices, querying whether it will ever be possible to completely do away with whole animal specimen collection.

Late Cenozoic history and the role of Beringia in assembling a Holarctic cestode species complex.

The dynamic climate history that drove sea level fluctuation during past glacial periods mediated the movement of organisms between Asia and North America via the Bering Land Bridge. Investigations of the biogeographic histories of small mammals and their parasites demonstrate facets of a complex history of episodic geographic colonization and refugial isolation that structured diversity across the Holarctic. We use a large multi-locus nuclear DNA sequence dataset to robustly resolve relationships within the cestode genus Arostrilepis (Cyclophyllidea: Hymenolepididae), a widespread parasite of predominantly arvicoline rodents (voles, lemmings). Using this phylogeny, we confirm that several Asian Arostrilepis lineages colonized North America during up to four distinct glacial periods in association with different rodent hosts, consistent with taxon-pulse dynamics. A previously inferred westward dispersal across the land bridge is rejected. We also refine interpretations of past host colonization, providing evidence for several distinct episodes of expanding host range, which probably contributed to diversification by Arostrilepis. Finally, Arostrilepis is shown to be paraphyletic with respect to Hymenandrya thomomyis, a parasite of pocket gophers, confirming that ancient Arostrilepis species colonized new host lineages upon arriving in North America.

SNP-based phylogenomic inference in Holarctic ground squirrels (Urocitellus).

Resolution of rapid evolutionary radiatons requires harvesting maximal signal from phylogenomic datasets. However, studies of non-model clades often target conserved loci that are characterized by reduced information content, which can negatively affect gene tree precision and species tree accuracy. Single nucleotide polymorphism (SNP)-based methods are an underutilized but potentially valuable tool for estimating phylogeny and divergence times because they do not rely on resolved gene trees, allowing information from many or all variant loci to be leveraged in species tree reconstruction. We evaluated the utility of SNP-based methods in resolving phylogeny of Holarctic ground squirrels (Urocitellus), a radiation that has been difficult to disentangle, even in prior phylogenomic studies. We inferred phylogeny from a dataset of >3,000 ultraconserved element loci (UCEs) using two methods (SNAPP, SVDquartets) and compared our results with a new mitogenome phylogeny. We also systematically evaluated how phasing of UCEs improves per-locus information content, inference of topology, and other parameters within each of these SNP-based methods. Phasing improved topological resolution and branch length estimation at shallow levels (within species complexes), but less so at deeper levels, likely reflecting true uncertainty due to ancestral polymorphisms segregating in rapidly diverging lineages. We resolved key clades in Urocitellus and present targeted opportunities for future phylogenomic inquiry. Our results also extend the roadmap for use of SNPs to address vertebrate radiations and inform comparative analyses at multiple temporal scales.

Disentangling lousy relationships: Comparative phylogenomics of two sucking louse lineages parasitizing chipmunks.

The evolution of obligate parasites is often interpreted in light of their hosts' evolutionary history. An expanded approach is to examine the histories of multiple lineages of parasites that inhabit similar environments on a particular host lineage. Western North American chipmunks (genus Tamias) have a broad distribution, a history of divergence with gene flow, and host two species of sucking lice (Anoplura), Hoplopleura arboricola and Neohaematopinus pacificus. From total genomic sequencing, we obtained sequences of over 1100 loci sampled across the genomes of these lice to compare their evolutionary histories and examine the roles of host association in structuring louse relationships. Within each louse species, clades are largely associated with closely related chipmunk host species. Exceptions to this pattern appear to have a biogeographic component, but differ between the two louse species. Phylogenetic relationships among these major louse clades, in both species, are not congruent with chipmunk relationships. In the context of host associations, each louse lineage has a different evolutionary history, supporting the hypothesis that host-parasite assemblages vary both across the landscape and with the taxa under investigation. In addition, the louse Hoplopleura erratica (parasitizing the eastern Tamias striatus) is embedded within H. arboricola, rendering it paraphyletic. This phylogenetic result, together with comparable divergences within H. arboricola, indicate a need for taxonomic revision. Both host divergence and biogeographic components shape parasite diversification as demonstrated by the distinctive diversification patterns of these two independently evolving lineages that parasitize the same hosts.

Impacts of Inference Method and Data set Filtering on Phylogenomic Resolution in a Rapid Radiation of Ground Squirrels (Xerinae: Marmotini).

Phylogenomic data sets are illuminating many areas of the Tree of Life. However, the large size of these data sets alone may be insufficient to resolve problematic nodes in the most rapid evolutionary radiations, because inferences in zones of extraordinarily low phylogenetic signal can be sensitive to the model and method of inference, as well as the information content of loci employed. We used a data set of $>$3950 ultraconserved element (UCE) loci from a classic mammalian radiation, ground-dwelling squirrels of the tribe Marmotini (Sciuridae: Xerinae), to assess sensitivity of phylogenetic estimates to varying per-locus information content across four different inference methods (RAxML, ASTRAL, NJst, and SVDquartets). Persistent discordance was found in topology and bootstrap support between concatenation- and coalescent-based inferences; among methods within the coalescent framework; and within all methods in response to different filtering scenarios. Contrary to some recent empirical UCE-based studies, filtering by information content did not promote complete among-method concordance. Nevertheless, filtering did improve concordance relative to randomly selected locus sets, largely via improved consistency of two-step summary methods (particularly NJst) under conditions of higher average per-locus variation (and thus increasing gene tree precision). The benefits of phylogenomic data set filtering are variable among classes of inference methods and across different evolutionary scenarios, reiterating the complexities of resolving rapid radiations, even with robust taxon and character sampling.

Sympatric Parasites Have Similar Host-Associated, but Asynchronous, Patterns of Diversification.

Parasitism is a common symbiotic interaction across diverse natural systems. Using a comparative evolutionary approach, we investigated the contributions of both host phylogeny and abiotic factors toward diversification of phylogenetically independent endoparasites that inhabit essentially the same physical space. We tested for host-parasite and parasite-parasite phylogenetic concordance in western North American chipmunks (Rodentia: Sciuridae) and two distantly related species of pinworms (Nematoda: Oxyurida). Deep structure in molecular phylogenies revealed signals of host-associated divergence in both parasite species, while shallower phylogeographic structure varied between the two parasites. This suggests that although these parasites experienced similar landscapes and cyclic climate processes, temporally distinctive diversification events were associated with differences in the initiation of their association with host lineages. When climate cycles initiate diversification, partially congruent, but asynchronous, host-associated parasite phylogenies may emerge.

Phylogenomics from Whole Genome Sequences Using aTRAM.

Novel sequencing technologies are rapidly expanding the size of data sets that can be applied to phylogenetic studies. Currently the most commonly used phylogenomic approaches involve some form of genome reduction. While these approaches make assembling phylogenomic data sets more economical for organisms with large genomes, they reduce the genomic coverage and thereby the long-term utility of the data. Currently, for organisms with moderate to small genomes ($<$1000 Mbp) it is feasible to sequence the entire genome at modest coverage ($10-30\times$). Computational challenges for handling these large data sets can be alleviated by assembling targeted reads, rather than assembling the entire genome, to produce a phylogenomic data matrix. Here we demonstrate the use of automated Target Restricted Assembly Method (aTRAM) to assemble 1107 single-copy ortholog genes from whole genome sequencing of sucking lice (Anoplura) and out-groups. We developed a pipeline to extract exon sequences from the aTRAM assemblies by annotating them with respect to the original target protein. We aligned these protein sequences with the inferred amino acids and then performed phylogenetic analyses on both the concatenated matrix of genes and on each gene separately in a coalescent analysis. Finally, we tested the limits of successful assembly in aTRAM by assembling 100 genes from close- to distantly related taxa at high to low levels of coverage.Both the concatenated analysis and the coalescent-based analysis produced the same tree topology, which was consistent with previously published results and resolved weakly supported nodes. These results demonstrate that this approach is successful at developing phylogenomic data sets from raw genome sequencing reads. Further, we found that with coverages above $5-10\times$, aTRAM was successful at assembling 80-90% of the contigs for both close and distantly related taxa. As sequencing costs continue to decline, we expect full genome sequencing will become more feasible for a wider array of organisms, and aTRAM will enable mining of these genomic data sets for an extensive variety of applications, including phylogenomics. [aTRAM; gene assembly; genome sequencing; phylogenomics.].

Revision of widespread red squirrels (genus: Tamiasciurus) highlights the complexity of speciation within North American forests.

Integration of molecular methods, ecological modeling, and statistical hypothesis testing are increasing our understanding of differentiation within species and phylogenetic relationships among species by revealing environmental connections to evolutionary processes. Within mammals, novel diversity is being discovered and characterized as more complete geographic sampling is coupled with newer multi-disciplinary approaches. North American red squirrels exemplify a forest obligate genus whose species are monitored as indicators of forest ecosystem condition, yet phylogenetic relationships reflecting evolutionary history within this genus remain tentative. Through testing of competing systematic and niche-based divergence hypotheses, we recognize three species, Tamiasciurus douglasii, T. hudsonicus, and T. fremonti. Our data provide evidence of regional differences in evolutionary dynamics and continental gradients of complexity that are important both for future management and for investigating multiple pathways that can lead to the formation of new species.

Lightella neohaematopini: A new lineage of highly reduced endosymbionts coevolving with chipmunk lice of the genus Neohaematopinus.

Sucking lice (Anoplura) are known to have established symbiotic associations multiple times with different groups of bacteria as diverse as Enterobacteriales, Legionellales, and Neisseriales. This diversity, together with absence of a common coevolving symbiont (such as Buchnera, in aphids), indicates that sucking lice underwent a series of symbiont acquisitions, losses, and replacements. To better understand evolution and significance of louse symbionts, genomic and phylogenetic data are needed from a broader taxonomic diversity of lice and their symbiotic bacteria. In this study, we extend the known spectrum of the louse symbionts with a new lineage associated with Neohaematopinus pacificus, a louse species that commonly parasitizes North American chipmunks. The recent coevolutionary analysis showed that rather than a single species, these lice form a cluster of unique phylogenetic lineages specific to separate chipmunk species (or group of closely related species). Using metagenomic assemblies, we show that the lice harbor a bacterium which mirrors their phylogeny and displays traits typical for obligate mutualists. Phylogenetic analyses place this bacterium within Enterobacteriaceae on a long branch related to another louse symbiont, "Candidatus Puchtella pedicinophila." We propose for this symbiotic lineage the name "Candidatus Lightella neohaematopini." Based on the reconstruction of metabolic pathways, we suggest that like other louse symbionts, L. neohaematopini provides its host with at least some B vitamins. In addition, several samples harbored another symbiotic bacterium phylogenetically affiliated with the Neisseriales-related symbionts described previously from the lice Polyplax serrata and Hoplopleura acanthopus. Characterizing these bacteria further extend the known diversity of the symbiotic associations in lice and show unique complexity and dynamics of the system.

SUCKING LICE (PHTHIRAPTERA: ANOPLURA) PARASITIZING MONGOLIAN RODENTS WITH THE DESCRIPTION OF A NEW SPECIES OF HOPLOPLEURA FROM MOUNTAIN VOLES (ALTICOLA SPP.).

The sucking louse fauna associated with Mongolian mammals is inadequately known. We provide a list of 25 species of sucking lice recorded from Mongolian rodents including previously published records, and new records of specimens collected during an expedition to northwestern Mongolia in 2015. Hoplopleura inagakii Ono and Hasegawa and Polyplax cricetulis Chin are newly recorded from Mongolia and 2 new host associations in Mongolia are recorded for Hoplopleura acanthopus (Burmeister). We describe Hoplopleura altaiensis n. sp., from the Gobi Altai mountain vole, Alticola barakshin Bannikov (type host) with an additional specimen from Alticola strelzowi (Kastchenko) (Strelzow's mountain vole). Both sexes of the new species are illustrated with scanning electron micrographs and line drawings. We note small morphological differences in the shape of the female subgenital plate between specimens prepared for scanning electron microscopy versus those prepared for light microscopy following DNA extraction.

Genomic resolution of cryptic species diversity in chipmunks.

Discovery of cryptic species is essential to understand the process of speciation and assessing the impacts of anthropogenic stressors. Here, we used genomic data to test for cryptic species diversity within an ecologically well-known radiation of North American rodents, western chipmunks (Tamias). We assembled a de novo reference genome for a single species (Tamias minimus) combined with new and published targeted sequence-capture data for 21,551 autosomal and 493 X-linked loci sampled from 121 individuals spanning 22 species. We identified at least two cryptic lineages corresponding with an isolated subspecies of least chipmunk (T. minimus grisescens) and with a restricted subspecies of the yellow-pine chipmunk (Tamias amoenus cratericus) known only from around the extensive Craters of the Moon lava flow. Additional population-level sequence data revealed that the so-called Crater chipmunk is a distinct species that is abundant throughout the coniferous forests of southern Idaho. This cryptic lineage does not appear to be most closely related to the ecologically and phenotypically similar yellow-pine chipmunk but does show evidence for recurrent hybridization with this and other species.

Lost in a sagebrush sea: comparative genetic assessment of an isolated montane population of Tamias amoenus.

The montane sky islands of the Great Basin are characterized by unique, isolated habitats and communities that likely are vulnerable to extirpation with environmental change. A subspecies of yellow pine chipmunk, the Humboldt yellow pine chipmunk (Tamias amoenus celeris), is associated with the whitebark and limber pine forests of the Pine Forest Range (PFR) in Nevada. We sampled T. amoenus and least chipmunks (T. minimus) from the isolated PFR and compared genetic diversity between these populations and more "mainland" populations, including other subspecies of chipmunks. Given the high frequency of hybridization in Tamias, we tested for hybridization between T. amoenus and T. minimus in the PFR. We examined phylogenetic relationships, population divergence and diversity, and screened populations for a common pathogen, Borrelia hermsii, to gain insight into population health. We found T. amoenus of the PFR are closely related to T. amoenus in the Warner Mountains and Sierra Nevada, but maintain substantively lower genetic variation. Microsatellite analyses show PFR T. amoenus are highly genetically differentiated from other populations. In contrast, PFR T. minimus had higher genetic diversity that was comparable to the other T. minimus population we sampled. Pathogen screening revealed that T. amoenus carried higher pathogen loads than T. minimus in the PFR, although the prevalence of infection was similar to other Tamias populations. Our assessment of habitat associations suggests that the Humboldt yellow pine chipmunk almost entirely is restricted to the conifer systems of the PFR, while least chipmunks are prevalent in the other forests. Our work highlights the need for continued conservation and research efforts to identify how response to environmental change can be facilitated in isolated species and habitats.

Natural history collections are critical resources for contemporary and future studies of urban evolution.

Urban environments are among the fastest changing habitats on the planet, and this change has evolutionary implications for the organisms inhabiting them. Herein, we demonstrate that natural history collections are critical resources for urban evolution studies. The specimens housed in these collections provide great potential for diverse types of urban evolution research, and strategic deposition of specimens and other materials from contemporary studies will determine the resources and research questions available to future urban evolutionary biologists. As natural history collections are windows into the past, they provide a crucial historical timescale for urban evolution research. While the importance of museum collections for research is generally appreciated, their utility in the study of urban evolution has not been explicitly evaluated. Here, we: (a) demonstrate that museum collections can greatly enhance urban evolution studies, (b) review patterns of specimen use and deposition in the urban evolution literature, (c) analyze how urban versus rural and native versus nonnative vertebrate species are being deposited in museum collections, and (d) make recommendations to researchers, museum professionals, scientific journal editors, funding agencies, permitting agencies, and professional societies to improve archiving policies. Our analyses of recent urban evolution studies reveal that museum specimens can be used for diverse research questions, but they are used infrequently. Further, although nearly all studies we analyzed generated resources that could be deposited in natural history collections (e.g., collected specimens), a minority (12%) of studies actually did so. Depositing such resources in collections is crucial to allow the scientific community to verify, replicate, and/or re-visit prior research. Therefore, to ensure that adequate museum resources are available for future urban evolutionary biology research, the research community-from practicing biologists to funding agencies and professional societies-must make adjustments that prioritize the collection and deposition of urban specimens.

No evidence for phylosymbiosis in western chipmunk species.

Phylosymbiosis refers to a congruent pattern between the similarity of microbiomes of different species and the branching pattern of the host phylogeny. Phylosymbiosis has been detected in a variety of vertebrate and invertebrate hosts, but has only been assessed in geographically isolated populations. We tested for phylosymbiosis in eight (sub)species of western chipmunks with overlapping ranges and ecological niches; we used a nuclear (Acrosin) and a mitochondrial (CYTB) phylogenetic marker because there are many instances of mitochondrial introgression in chipmunks. We predicted that similarity among microbiomes increases with: (1) increasing host mitochondrial relatedness, (2) increasing host nuclear genome relatedness and (3) decreasing geographic distance among hosts. We did not find statistical evidence supporting phylosymbiosis in western chipmunks. Furthermore, in contrast to studies of other mammalian microbiomes, similarity of chipmunk microbiomes is not predominantly determined by host species. Sampling site explained most variation in microbiome composition, indicating an important role of local environment in shaping microbiomes. Fecal microbiomes of chipmunks were dominated by Bacteroidetes (72.2%), followed by Firmicutes (24.5%), which is one of the highest abundances of Bacteroidetes detected in wild mammals. Future work will need to elucidate the effects of habitat, ecology and host genomics on chipmunk microbiomes.

Building an integrated infrastructure for exploring biodiversity: field collections and archives of mammals and parasites.

Museum specimens play an increasingly important role in predicting the outcomes and revealing the consequences of anthropogenically driven disruption of the biosphere. As ecological communities respond to ongoing environmental change, host-parasite interactions are also altered. This shifting landscape of host-parasite associations creates opportunities for colonization of different hosts and emergence of new pathogens, with implications for wildlife conservation and management, public health, and other societal concerns. Integrated archives that document and preserve mammal specimens along with their communities of associated parasites and ancillary data provide a powerful resource for investigating, anticipating, and mitigating the epidemiological, ecological, and evolutionary impacts of environmental perturbation. Mammalogists who collect and archive mammal specimens have a unique opportunity to expand the scope and impact of their field work by collecting the parasites that are associated with their study organisms. We encourage mammalogists to embrace an integrated and holistic sampling paradigm and advocate for this to become standard practice for museum-based collecting. To this end, we provide a detailed, field-tested protocol to give mammalogists the tools to collect and preserve host and parasite materials that are of high quality and suitable for a range of potential downstream analyses (e.g., genetic, morphological). Finally, we also encourage increased global cooperation across taxonomic disciplines to build an integrated series of baselines and snapshots of the changing biosphere. Los especímenes de museo desempeñan un papel cada vez más importante tanto en la descripción de los resultados de la alteración antropogénica de la biosfera como en la predicción de sus consecuencias. Dado que las comunidades ecológicas responden al cambio ambiental, también se alteran las interacciones hospedador-parásito. Este panorama cambiante de asociaciones hospedador-parásito crea oportunidades para la colonización de diferentes hospedadores y para la aparición de nuevos patógenos, con implicancias en la conservación y manejo de la vida silvestre, la salud pública y otras preocupaciones de importancia para la sociedad. Archivos integrados que documentan y preservan especímenes de mamíferos junto con sus comunidades de parásitos y datos asociados, proporcionan un fuerte recurso para investigar, anticipar y mitigar los impactos epidemiológicos, ecológicos y evolutivos de las perturbaciones ambientales. Los mastozoólogos que recolectan y archivan muestras de mamíferos, tienen una oportunidad única de ampliar el alcance e impacto de su trabajo de campo mediante la recolección de los parásitos que están asociados con los organismos que estudian. Alentamos a los mastozoólogos a adoptar un paradigma de muestreo integrado y holístico y abogamos para que esto se convierta en una práctica estándarizada de la obtención de muestras para museos. Con este objetivo, proporcionamos un protocolo detallado y probado en el campo para brindar a los mastozoólogos las herramientas para recolectar y preservar materiales de parásitos y hospedadores de alta calidad y adecuados para una gran variedad de análisis subsecuentes (e.g., genéticos, morfológicos, etc.). Finalmente, también abogamos por una mayor cooperación global entre las diversas disciplinas taxonómicas para construir una serie integrada de líneas de base y registros actuales de nuestra cambiante biosfera.

Parasite Collections: Overlooked Resources for Integrative Research and Conservation.

Parasite natural history collections form vital scientific infrastructure that play a substantial role in increasing awareness of the importance of parasites to ecosystems, conservation assessments, science, and society. These collections support novel investigations that integrate across taxa, time, and space, and should be cultivated to advance organismal-based science. Promoting and supporting parasite collections will ensure their ongoing stability and accessibility.

Temporal and spatial mosaics: deep host association and shallow geographic drivers shape genetic structure in a widespread pinworm, Rauschtineria eutamii.

Climate and host demographic cycling often shape both parasite genetic diversity and host distributions, processes that transcend a history of strict host-parasite association. We explored host associations and histories based on an evaluation of mitochondrial and nuclear sequences to reveal the underlying history and genetic structure of a pinworm, Rauschtineria eutamii, infecting 10 species of western North American chipmunks (Rodentia:Tamias, subgenus Neotamias). Rauschtineria eutamii contains divergent lineages influenced by the diversity of hosts and variation across the complex topography of western North America. We recovered six reciprocally monophyletic R. eutamii mitochondrial clades, largely supported by nuclear gene trees, exhibiting divergence levels comparable to intraspecific variation reported for other nematodes. Phylogenetic relationships among pinworm clades suggest that R. eutamii colonized an ancestral lineage of western chipmunks and lineages persisted during historical isolation in diverging Neotamias species or species groups. Pinworm diversification, however, is incongruent and asynchronous relative to host diversification. Secondarily, patterns of shallow divergence were shaped by geography through events of episodic colonization reflecting an interaction of taxon pulses and ecological fitting among assemblages in recurrent sympatry. Pinworms occasionally infect geographically proximal host species; however, host switching may be unstable or ephemeral, as there is no signal of host switching in the deeper history of R. eutamii.

Natural history collections-based research: progress, promise, and best practices.

Specimens and associated data in natural history collections (NHCs) foster substantial scientific progress. In this paper, we explore recent contributions of NHCs to the study of systematics and biogeography, genomics, morphology, stable isotope ecology, and parasites and pathogens of mammals. To begin to assess the magnitude and scope of these contributions, we analyzed publications in the Journal of Mammalogy over the last decade, as well as recent research supported by a single university mammal collection (Museum of Southwestern Biology, Division of Mammals). Using these datasets, we also identify weak links that may be hindering the development of crucial NHC infrastructure. Maintaining the vitality and growth of this foundation of mammalogy depends on broader engagement and support from across the scientific community and is both an ethical and scientific imperative given the rapidly changing environmental conditions on our planet.