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Bird nests as botanical time capsules: DNA barcoding identifies the contents of contemporary and historical nests

['Alex Rinkert', 'Department Of Biological Sciences', 'San José State University', 'San José', 'Ca', 'United States Of America', 'Tracy M. Misiewicz', 'Department Of Microbiology', 'Plant Biology', 'University Of Oklahoma']

Date: 2021-11

Abstract Bird nests in natural history collections are an abundant yet vastly underutilized source of genetic information. We sequenced the nuclear ribosomal internal transcribed spacer to identify plant species used as nest material in two contemporary (2003 and 2018) and two historical (both 1915) nest specimens constructed by Song Sparrows (Melospiza melodia) and Savannah Sparrows (Passerculus sandwichensis). A total of 13 (22%) samples yielded single, strong bands that could be identified using GenBank resources: six plants (Angiospermae), six green algae (Chlorophyta), and one ciliate (Ciliophora). Two native plant species identified in the nests included Festuca microstachys, which was introduced to the nest collection site by restoration practitioners, and Rosa californica, identified in a nest collected from a lost habitat that existed about 100 years ago. Successful sequencing was correlated with higher sample mass and DNA quality, suggesting future studies should select larger pieces of contiguous material from nests and materials that appear to have been fresh when incorporated into the nest. This molecular approach was used to distinguish plant species that were not visually identifiable, and did not require disassembling the nest specimens as is a traditional practice with nest material studies. The many thousands of nest specimens in natural history collections hold great promise as sources of genetic information to address myriad ecological questions.

Citation: Rinkert A, Misiewicz TM, Carter BE, Salmaan A, Whittall JB (2021) Bird nests as botanical time capsules: DNA barcoding identifies the contents of contemporary and historical nests. PLoS ONE 16(10): e0257624. https://doi.org/10.1371/journal.pone.0257624 Editor: Hideyuki Doi, University of Hyogo, JAPAN Received: April 21, 2021; Accepted: September 6, 2021; Published: October 6, 2021 Copyright: © 2021 Rinkert et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: AR received a grant from Northern California Botanists (http://www.norcalbotanists.org/). AR and JBW received a grant from Midpeninsula Regional Open Space (https://www.openspace.org/). Additional funding was provided by Santa Clara Valley Water District (https://www.valleywater.org/) and San Francisco Bay Bird Observatory (https://www.sfbbo.org/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Bird nest specimens are abundant in many natural history collections, yet they are vastly underutilized as a tool for answering questions in ecology [1]. Worldwide there are nearly 60,000 bird nest specimens archived in natural history collections that span the past 250 years [2, 3]. As new technologies emerge, museum specimens are increasingly being used as a source of genetic information [4]. The spatial, temporal, and taxonomic representation of nest specimens makes them an attractive and untapped source of ecological data [1], and nest material can be used to study a variety of ecological topics such as architectural camouflage, chemical defense from pathogens and parasites, intraspecific signaling, seed dispersal, and mammal biogeography [5–11]. The increasing ubiquity of artificial materials, especially plastics, in the nests of some species has also demonstrated the utility of learning about pollution and land use change in the environment through materials incorporated into bird nests (see [12]). Despite many birds using plants as nest material, few studies attempt to make inferences from nests made primarily of plant material, possibly because of the challenges associated with identifying the plant species from this material type. Identifying plant material in bird nests is hindered because pieces of plant material often do not contain taxonomically useful and distinctive structures. The number of plant species and amount of materials that can be identified varies tremendously (see [6, 13–15]), and the distinctiveness of the plant material incorporated into a nest undoubtedly determines how much material in the nest can be identified. For example, many passerines, such as New World sparrows (Emberizidae), use dried grasses as nest material [16]. This plant material typically does not contain the reproductive structures that species identifications hinge upon, rendering the principal material in these nests visually unidentifiable. The age of nests that can be studied is a further limitation, as while studies describing the types of materials in freshly collected nests are typically disassembled and examined ex situ (e.g., [8, 12, 15]), this destructive sampling practice is avoided in natural history collections (e.g., [5, 17]) likely because of the historical value of the specimens. A number of advances in molecular biology have facilitated the genetic analysis of historical specimens. Next generation sequencing technologies are compatible with fragmented and degraded DNA that is typical of museum specimens [18, 19] and the development of primers that target short amplicons have led to successful species identifications from degraded plant material, including ancient material dating back 10,000 years [20, 21]. The techniques used to obtain DNA from preserved plant specimens have overcome various challenges with specimen preparation and taxon-specific traits (see [22]), as well as restrictions for destructively sampling historical specimens [23]. Some of these challenges are no different than those that would be encountered with bird nest specimens, albeit the quality of the materials may be poorer as nests are often made of dead plant materials, unlike plant specimens which are typically collected from a living organism. We investigated the utility of using the nuclear ribosomal internal transcribed spacer (ITS) barcode [24] to identify plant species from bird nest material. We identified several plant species from bird nests and identified sample characteristics that can be used as predictors for sequencing success. Finally, we provide recommendations for using a molecular approach to identify plant species from nest material.

Discussion Genetic information from bird nests Thirteen samples representing historical and contemporary collection eras were successfully sequenced from bird nest specimens, and sequences could be identified using existing references in GenBank. While most samples lacked recognizable structures that could be used to visually identify the plant species, by using a molecular approach we identified six plants that likely represent five species. Specimens in natural history collections often serve as a source of genetic information to answer ecological and evolutionary questions (e.g,. [18, 19]), however we are unaware of plant material from bird nest specimens previously being used for this purpose. Revealing the historical ecology of lost habitats Nest specimens are botanical time capsules containing a physical representation of the environment where they were collected. Plants identified in historical nests can unlock new information about the ecology of historical environments that have since changed. We identified a native rose (Rosa) in an historical nest (MVZ1:Egg:1613) collected in a habitat that has since been lost to dramatic landscape changes. While some Song Sparrows are known to use Rosa shrubs as a nest substrate [35], the R. californica sample selected from the nest specimen was a fine stem woven between the lining and structure so it is unlikely to be part of the nest substrate but rather intentionally incorporated into the nest. Furthermore, no other apparent Rosa material was noted in the nest. The historical nest specimen was constructed by a tidal marsh-dwelling Song Sparrow, M. m. maxillaris, which nests in marshes adjacent to transitional habitat in the San Francisco Bay estuary [36]. The nests of this taxon are lined with grasses and contain other plant material that does not grow in tidally influenced marshes where they nest, but rather originates from the adjacent transitional habitat (A. Rinkert pers. obs.). Transitional habitat, the ecological gradient between the tidal marshes and uplands [37], was lost by the early 1900s as a result of major landscape changes [38, 39], and consequently, very little is known about the former native plant community. The only historical description of the plant community in transitional habitat [38] mentioned ten plant species, including R. californica, growing in relict patches between 1913–1915. The R. california identified in the historical nest specimen confirms this plant species was historically part of transitional habitat. Further sampling and sequencing from historical nest specimens of Song Sparrows from the San Francisco Bay estuary could reconstruct transitional habitat more completely by revealing additional plant species once occurring there. While the genetic identity and phylogenetic analyses support the identification as R. californica (S7 Fig), the genus Rosa is incompletely represented in GenBank. Only two species of native California Rosa—R. californica and R. woodsii—are represented by ITS sequences in GenBank (GenBank Release 241: December 15 2020). Furthermore, R. californica is apparently weakly differentiated at the trnL chloroplast loci from R. nutkana [40], another native California Rosa that is known to occur near marshes in the estuary and does not have an ITS reference sequence in GenBank [41, 42]. Incomplete representation of species in GenBank may prove to be an initial limitation to identifying species from nest materials, however the existing GenBank resources were able to identify all six plant species to genus, and species identifications are possible with the development of reference sequences in target genera (S1 Protocol). Bird nests as a tool for restoration Nest material can be used to help determine the success of habitat restoration. Restoration success of a native plant community is rarely evaluated, especially on a holistic scale [43]. We identified Festuca microstachys, a California native [44] incorporated into a nest specimen collected in a habitat restoration area at Bair Island, CA (Table 2, S5 Fig). This restoration area was formerly a decommissioned salt pond with narrow levee slopes covered in exotic vegetation. Prior to restoration at this site in 2016, there were no occurrences of F. microstachys at Bair Island in plant observation databases [42, 45] and the only native species observed on these levee slopes prior to restoration were Baccharis pilularis and Salicornia pacifica (A. Rinkert pers. obs.). In 2017 restoration practitioners introduced a broad suite of native species, including F. microstachys, to this site to establish a native plant community [46, 47; S2 Table]. The nest specimen SFBBO2 was subsequently collected 2 m from the restoration area in 2018. Considering the apparent absence of F. microstachys prior to restoration, we conclude this nest material originated from a plant introduced by restoration practitioners, which demonstrates birds immediately utilize native plant species for nest building. We propose comparing the nest materials of birds before and after restoration to evaluate the recovery of ecological interactions, as well as determine plant species preferences for nest material. Restoration areas typically offer a broad suite of native plants which, combined with vegetation surveys at the site, can offer insight into the selectiveness of certain plants for nest materials, further refining restoration efforts in the future to better benefit species that select specific materials for their nest. Circumventing challenges with studying nest material A major advantage of using a molecular approach to identify plants in bird nests is that the materials do not need to retain any structures that could be used to visually identify the species. Two nondescript pieces of grass culms were identified to the tribe Triticeae, with the likely species being Elymus triticoides (S5 Fig, Table 2), which is native to the collection site [44]. The bioinformatic analyses could not distinguish the two sample sequences from two other species (E. ambiguus and E. cinereus) within Elymus, but they are either unrecorded in California or occur very rarely in the San Francisco Bay estuary [42, 48], so we conclude these two samples were E. triticoides, a common species at the collection site [44, A. Rinkert pers. obs.]. The taxonomic resolution of this species identification would not be possible to achieve with visual examination as the materials do not retain structures that are used to distinguish these three species in Elymus [48]. A broader range of nest types could be sampled by using a molecular approach as species identification would not hinge on the material retaining identifiable structures, and the nest specimen would not need to be disassembled in contrast with traditional practices. Considerations for sanger sequencing of nest material Utilizing historical and contemporary nest specimens. Historical nest specimens in natural history collections are an invaluable source of genetic information, however the preservation conditions of these historical specimens warrant consideration when sampling. The preservation technique and storage of many historical nest specimens may be unknown. Cross-contamination between specimens stored in the same physical space may have occurred, and for this reason we recommend only selecting materials that are incorporated into the architecture of the nest, and not loose on the exterior of the nest or detached materials accompanying the specimen. Historical nest specimens were also typically subjected or inadvertently exposed to various types of chemical treatments (see [49]). These chemicals could interfere with the molecular processes used to obtain DNA from historical nest specimens (e.g., [50, 51]). When possible, we recommend choosing specimens to sample from that are known to not have been treated with chemicals as a precautionary measure. The same considerations for historical nest specimens generally apply to contemporary nest. Studies calling for the collection of nest specimens should store nests separately in sterile containers and the nests should not be chemically treated. Instead, nests should be frozen or dried, both of which preserve DNA in woody plant material [52]. Lastly, identifying the species or type of nest substrate can help with interpreting species identifications from the nest. If materials from the nest substrate are inadvertently sampled and identified, these results should be interpreted cautiously as the materials may not have been deliberately added to the nest. Identifying appropriate specimens and materials for sampling. Nests that are made with degraded plant material introduce challenges with obtaining high quality sequences as low yields and environmentally caused damage and contamination can interfere with molecular procedures, sharing similar challenges with ancient DNA [53, 54]. We recommend larger pieces of contiguous material be selected from nest specimens to maximize the yield of DNA obtained; if no larger pieces of material exist, then multiple samples of similar morphology can be pooled to obtain an equivalent critical mass. Higher dry sample mass and DNA concentration were predictors of sequencing success, likely because of the proportional increase in DNA yield with mass. Selecting specific pieces of material based on their morphological or visual characteristics may also lead to higher sequencing success. Only one green leaf was observed in the four nests we sampled, and the sample of this material yielded the Rosa californica identification. While green leaves have little effect on the DNA obtained from historical herbarium specimens [55], the leaf from the nest specimen may have been freshly collected and more quickly desiccated, and therefore allowing the green color and DNA to be better preserved. Leaves are ubiquitously used as a source of DNA for botanical studies [52] making this material in nests most advantageous to sample, however new techniques can help overcome some of the physical and chemical challenges with obtaining DNA from woody stems and aromatic material, that are sometimes used by birds as nest material. Selecting specific portions of woody stems and ensuring complete desiccation can maximize the yield of DNA in woody stems [52], while modifying standard CTAB extraction protocols to better handle undesirable chemical compounds can improve DNA quality [56]. Sanger sequencing may be most effective on nest specimens made of certain types of materials. Nests constructed of green, fresh material may produce samples with higher yields of DNA and resultantly higher sequencing success, as this material is most similar to what is conventionally used in botanical studies [52]. Species such as the European Starling (Sturnus vulgaris), weaverbirds (Ploceidae), and some swifts (Cypseloidinae) collect fresh nest material from living plants, and in some cases build a nest that is a living organism itself [9, 57–59], making these ideal nest types for studies utilizing Sanger sequencing. Identifying a genetic marker. Considering over 90% of candidate plant species from the nest collection sites were represented in GenBank (S2 Table), we suggest this approach has widespread utility in other comparably sampled habitats. While GenBank serves as an adequate reference library, housing over 379,637 non-redundant angiosperm sequences (GenBank Release 241: accessed February 15 2021), confident species identification using ITS alone can be limited by several factors. The lack of sequence variation among closely related species, inconsistent phylogenetic signals at the tips of the tree (e.g., Elymus), and incomplete sampling of taxonomic groups all limit the taxonomic certainty and resolution of identification. For the latter, we demonstrate with R. californica that even if the taxonomic group is incompletely sampled, GenBank resources provide an approximate identification (e.g., to genus) that can be narrowed when supplemented with reference sequences from herbarium specimens or fresh field collections, such as R. californica (e.g., S1 Protocol). Including additional, rapidly evolving and widely sampled loci such as cpDNA introns/spacers or nuclear introns from conserved orthologous genes [60–62] may improve confidence in species identifications. The high percentage of plant species at the collection sites with ITS reference in GenBank helps assure species identifications are accurate, and should be considered as a prerequisite for selecting nest specimens to sample. Distinguishing between target and spurious results. Spurious results may be produced when using a highly sensitive molecular procedure to amplify extremely low yields of DNA (see [63, 64]). While we targeted the kingdom Plantae, we amplified and identified six taxa of green algae and one ciliate (Table 2). The five green algae sequences we could identify to species using BLAST are multicellular organisms known to occur in marine and brackish water [65–68], while the one ciliate identified is also known to be a marine species [69]. While none of the green algae are specifically known to occur in the San Francisco Bay estuary, where all four nest specimens were collected, it is plausible they are naturalized as the marine ecosystem in this estuary is known to be highly invaded [70]. All green algae sequences had high BLAST E-values and all but two (samples 12–10 and 17–8) represent different lineages [65–68]. We postulate these green algae and the ciliate were incidentally deposited on the nest material as all nest specimens were collected from areas with some tidal influence or stream flow, instead of originating from lab contamination. Future directions Molecularly identifying plants in nest specimens is a viable method of identifying species, however the type of nest material and overarching goals of the study will determine whether the Sanger sequencing approach is appropriate. Sampling across a wider variety of nest types will further ascertain what species build nests that are most conducive to obtaining DNA through this method, and whether climate and nest material type factor into the preservation of the materials. There is a wealth of ecological and evolutionary questions that can be addressed with DNA obtained from nest specimens, especially with the temporal and spatial breadth of specimens available in natural history collections. Sampling historical nest specimens in conjunction with contemporary specimens can be used to determine how nest material preferences have changed following land use changes, and can even identify shifts in nest material preference as a result of climate change [71]. For some species, preferences for specific types of nest materials differ across their range [57, 72]. Further ascertaining geographical differences in nest material preferences may reveal patterns corresponding with the relative availability of specific types of nest materials, and perhaps identify whether the availability of these materials may limit their distribution. The nest material used by some species may not vary spatially or temporally, and identifying and understanding the evolution of these strong preferences may further help conservation efforts. Beyond plants, other communities could even be reconstructed from DNA obtained from nest specimens. Invertebrate and mammal remains identified in contemporary bird nests have confirmed the presence of species in areas where they were previously not known to occur (e.g., [8, 73]). Finally, the application of third generation genome sequencing technologies applied to new materials like bird nests will greatly expand the prospects of identifying ecological connections in past and present ecosystems.

Acknowledgments We thank Douglas G. D. Russell and one anonymous reviewer for their thoughtful and constructive feedback that greatly improved this manuscript. Carla Cicero kindly provided access to the Museum of Vertebrate Zoology collections at the University of California, Berkeley. Santa Clara University Biology staff provided essential lab and computer support.

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