Performant barcode decoding for herbarium specimen images using vector-assisted region proposals (VARP).

PREMISE: The scale and associated costs of herbarium digitization make process automation appealing. One such process for many workflows is the association of specimen image files with barcode values stored with the specimen. Here, an innovation is presented that improves the speed and accuracy of decoding barcodes from specimen images. METHODS AND RESULTS: Geometric features common in barcodes are used to identify the regions of specimen images that are likely to contain a barcode. The proposed regions are then combined into a significantly reduced composite image that is decoded using traditional barcode reading libraries. Tested against existing solutions, this method demonstrated the highest success rate (96.5%) and the second fastest processing time (617 ms). CONCLUSIONS: This method was developed to support a larger effort to automate specimen image post-processing in real-time, highlighting the importance of execution time. Although initially designed for herbarium digitization, this method may be useful for other high-resolution applications.

Estimating herbarium specimen digitization rates: Accounting for human experience.

PREMISE: Herbaria are invaluable sources for understanding the natural world, and in recent years there has been a concerted effort to digitize these collections. To organize such efforts, a method for estimating the necessary labor is desired. This work analyzes digitization productivity reports of 105 participants from eight herbaria, deriving generalized labor estimates that account for human experience. METHODS AND RESULTS: Individuals' rates of digitization were grouped based on cumulative time performing each task and then used to estimate a series of generalized labor projection models. In most cases, productivity was shown to improve with experience, suggesting longer technician retention can reduce labor requirements by 20%. CONCLUSIONS: Using student labor is a common tactic for digitization efforts, and the resulting outreach exposes future professionals to natural history collections. However, overcoming the learning curve should be considered when estimating the labor necessary to digitize a collection.

Enabling automated herbarium sheet image post-processing using neural network models for color reference chart detection.

PREMISE: Large-scale efforts to digitize herbaria have resulted in more than 18 million publicly available Plantae images on sites such as iDigBio. The automation of image post-processing will lead to time savings in the digitization of biological specimens, as well as improvements in data quality. Here, new and modified neural network methodologies were developed to automatically detect color reference charts (CRC), enabling the future automation of various post-processing tasks. METHODS AND RESULTS: We used 1000 herbarium specimen images from 52 herbaria to test our novel neural network model, ColorNet, which was developed to identify CRCs smaller than 4 cm2, resulting in a 30% increase in accuracy over the performance of other state-of-the-art models such as Faster R-CNN. For larger CRCs, we propose modifications to Faster R-CNN to increase inference speed. CONCLUSIONS: Our proposed neural networks detect a range of CRCs, which may enable the automation of post-processing tasks found in herbarium digitization workflows, such as image orientation or white balance correction.

A born-digital field-to-database solution for collections-based research using collNotes and collBook.

PREMISE: The digitization of natural history collections includes transcribing specimen label data into standardized formats. Born-digital specimen data initially gathered in digital formats do not need to be transcribed, enabling their efficient integration into digitized collections. Modernizing field collection methods for born-digital workflows requires the development of new tools and processes. METHODS AND RESULTS: collNotes, a mobile application, was developed for Android and iOS to supplement traditional field journals. Designed for efficiency in the field, collNotes avoids redundant data entries and does not require cellular service. collBook, a companion desktop application, refines field notes into database-ready formats and produces specimen labels. CONCLUSIONS: collNotes and collBook can be used in combination as a field-to-database solution for gathering born-digital voucher specimen data for plants and fungi. Both programs are open source and use common file types simplifying either program's integration into existing workflows.

A rangewide herbarium-derived dataset indicates high levels of gene flow in black cherry (Prunus serotina).

Isolation by Distance (IBD) is a genetic pattern in which populations geographically closer to one another are more genetically similar to each other than populations which are farther apart. Black cherry (Prunus serotina Ehrh.) (Rosaceae) is a forest tree species widespread in eastern North America, and found sporadically in the southwestern United States, Mexico, and Guatemala. IBD has been studied in relatively few North American plant taxa, and no study has rigorously sampled across the range of such a widespread species. In this study, IBD and overall genetic structure were assessed in eastern black cherry (P. serotina Ehrh. var. serotina), the widespread variety of eastern North America. Eastern North America. Prunus serotina Ehrh. var. serotina (Rosaceae). Dense sampling across the entire range of eastern black cherry was made possible by genotyping 15 microsatellite loci in 439 herbarium samples from all portions of the range. Mantel tests and STRUCTURE analyses were performed to evaluate the hypothesis of IBD and genetic structure. Mantel tests demonstrated significant but weak IBD, while STRUCTURE analyses revealed no clear geographic pattern of genetic groups. The modest geographic/genetic structure across the eastern black cherry range suggests widespread gene flow in this taxon. This is consistent with P. serotina's status as a disturbance-associated species. Further studies should similarly evaluate IBD in species characteristic of low-disturbance forests.

Markers in time and space: A review of the last decade of plant phylogeographic approaches.

Plant studies comprise a relatively small proportion of the phylogeographic literature, likely as a consequence of the fundamental challenges posed by the complex genomic structures and life history strategies of these organisms. Comparative plastomics (i.e., comparisons of mutation rates within and among regions of the chloroplast genome) across plant lineages has led to an increased understanding of which markers are likely to provide the most information at low taxonomic levels. However, the extent to which the results of such work have influenced the literature has not been fully assessed, nor has the extent to which plant phylogeographers explicitly analyse markers in time and space, both of which are integral components of the field. Here, we reviewed more than 400 publications from the last decade of plant phylogeography to specifically address the following questions: (i) What is the phylogenetic breadth of studies to date? (ii) What molecular markers have been used, and why were they chosen? (iii) What kinds of markers are most frequently used and in what combinations? (iv) How frequently are divergence time estimation and ecological niche modelling used in plant phylogeography? Our results indicate that chloroplast DNA sequence data remain the primary tool of choice, followed distantly by nuclear DNA sequences and microsatellites. Less than half (42%) of all studies use divergence time estimation, while even fewer use ecological niche modelling (14%). We discuss the implications of our findings, as well as the need for community standards on data reporting.

Digitization workflows for flat sheets and packets of plants, algae, and fungi.

Effective workflows are essential components in the digitization of biodiversity specimen collections. To date, no comprehensive, community-vetted workflows have been published for digitizing flat sheets and packets of plants, algae, and fungi, even though latest estimates suggest that only 33% of herbarium specimens have been digitally transcribed, 54% of herbaria use a specimen database, and 24% are imaging specimens. In 2012, iDigBio, the U.S. National Science Foundation's (NSF) coordinating center and national resource for the digitization of public, nonfederal U.S. collections, launched several working groups to address this deficiency. Here, we report the development of 14 workflow modules with 7-36 tasks each. These workflows represent the combined work of approximately 35 curators, directors, and collections managers representing more than 30 herbaria, including 15 NSF-supported plant-related Thematic Collections Networks and collaboratives. The workflows are provided for download as Portable Document Format (PDF) and Microsoft Word files. Customization of these workflows for specific institutional implementation is encouraged.

Chloroplast DNA sequence utility for the lowest phylogenetic and phylogeographic inferences in angiosperms: the tortoise and the hare IV.

PREMISE OF THE STUDY: Noncoding chloroplast DNA (NC-cpDNA) sequences are the staple data source of low-level phylogeographic and phylogenetic studies of angiosperms. We followed up on previous papers (tortoise and hare II and III) that sought to identify the most consistently variable regions of NC-cpDNA. We used an exhaustive literature review and newly available whole plastome data to assess applicability of previous conclusions at low taxonomic levels. METHODS: We aligned complete plastomes of 25 species pairs from across angiosperms, comparing the number of genetic differences found in 107 NC-cpDNA regions and matK. We surveyed Web of Science for the plant phylogeographic literature between 2007 and 2013 to assess how NC-cpDNA has been used at the intraspecific level. KEY RESULTS: Several regions are consistently the most variable across angiosperm lineages: ndhF-rpl32, rpl32-trnL((UAG)), ndhC-trnV((UAC)), 5'rps16-trnQ((UUG)), psbE-petL, trnT((GGU))-psbD, petA-psbJ, and rpl16 intron. However, there is no universally best region. The average number of regions applied to low-level studies is ∼2.5, which may be too little to access the full discriminating power of this genome. CONCLUSIONS: Plastome sequences have been used successfully at lower and lower taxonomic levels. Our findings corroborate earlier works, suggesting that there are regions that are most likely to be the most variable. However, while NC-cpDNA sequences are commonly used in plant phylogeographic studies, few of the most variable regions are applied in that context. Furthermore, it appears that in most studies too few NC-cpDNAs are used to access the discriminating power of the cpDNA genome.

Diversification of almonds, peaches, plums and cherries - molecular systematics and biogeographic history of Prunus (Rosaceae).

Most previous molecular phylogenetic studies of Prunus have been conducted primarily with crop species and their close relatives. As the center of crop diversity of the genus is in Eurasia, the geographic origin of Prunus has inevitably been inferred to be Eurasia as well. The lesser-known tropical Prunus species have not been well represented in previous phylogenetic reconstructions; therefore, their effects on inferences about the phylogenetic structure and geographic origin of Prunus are uncertain. In this study, we examined the phylogeny of Prunus, including an expanded sampling of species from tropical regions in Southeast Asia and the Americas, using sequences from four plastid markers and the nuclear ribosomal ITS region. A penalized likelihood method was used to estimate the absolute age of Prunus and the timing of infrageneric cladogenic events. The geographic origin of Prunus and ancestral sites of cladogenesis were inferred using the Bayes-DIVA approach. Our results indicate that the modern genus appeared ∼61Myr in eastern Asia and that diversification of all major lineages may have been triggered by the global warming period of the early Eocene. In addition, our molecular dating estimates suggest that the crown clade that includes the temperate deciduous crop species is older than the one that includes the tropical evergreen species, while incongruence between plastid and nuclear phylogenies suggests that the latter lineage originated via an ancient hybridization event. The most recent common ancestor (MRCA) of the temperate crop species was a component of the continuous boreotropical forests of the Northern Hemisphere, while the MRCA of the tropical species represented the last remains of the boreotropical elements and subsequently radiated throughout the Old and New World tropics from refugial areas at lower latitudes. Complex biogeographic histories leading to the present global distribution of the genus were driven by several geologic events, climatic oscillations, and independent dispersals across continents via the Bering and the North Atlantic Land Bridges during different geologic time periods.

Hydraulic management of a soil moisture controlled SDI wastewater dispersal system in an Alabama Black Belt soil.

Rural areas represent approximately 95% of the 14000 km(2) Alabama Black Belt, an area of widespread Vertisols dominated by clayey, smectitic, shrink-swell soils. These soils are unsuitable for conventional onsite wastewater treatment systems (OWTS) which are nevertheless widely used in this region. In order to provide an alternative wastewater dosing system, an experimental field moisture controlled subsurface drip irrigation (SDI) system was designed and installed as a field trial. The experimental system that integrates a seasonal cropping system was evaluated for two years on a 500-m(2) Houston clay site in west central Alabama from August 2006 to June 2008. The SDI system was designed to start hydraulic dosing only when field moisture was below field capacity. Hydraulic dosing rates fluctuated as expected with higher dosing rates during warm seasons with near zero or zero dosing rates during cold seasons. Lower hydraulic dosing in winter creates the need for at least a two-month waste storage structure which is an insurmountable challenge for rural homeowners. An estimated 30% of dosed water percolated below 45-cm depth during the first summer which included a 30-year historic drought. This massive volume of percolation was presumably the result of preferential flow stimulated by dry weather clay soil cracking. Although water percolation is necessary for OWTS, this massive water percolation loss indicated that this experimental system is not able to effective control soil moisture within its monitoring zone as designed. Overall findings of this study indicated that soil moisture controlled SDI wastewater dosing is not suitable as a standalone system in these Vertisols. However, the experimental soil moisture control system functioned as designed, demonstrating that soil moisture controlled SDI wastewater dosing may find application as a supplement to other wastewater disposal methods that can function during cold seasons.

Population dynamics and genetic variability of Escherichia coli in a mixed land-use watershed.

Better understanding of Escherichia coli population dynamics and genetic variability in the secondary habitat is essential to improve fecal contamination monitoring and contamination pathway characterization. In this study, water samples were collected monthly over a one-year period at eight locations in the Catoma Creek watershed, a mixed land-use watershed in Central Alabama. E. coli concentrations varied from 17 to 12,650 CFU/100 ml and were well correlated with stream flow rates. Repetitive sequence-based PCR DNA fingerprinting was used to generate 271 unique DNA fingerprint patterns from 502 E. coli isolated from water samples. Cluster analysis showed an overall similarity of 32.8% across all DNA fingerprints. Multivariate analysis of variance (MANOVA) showed that E. coli genotypes had a tendency to cluster according to season and stream flow rather than sampling sites. MANOVA of a subset of data within a given season and flow rate, however, revealed some geographical differentiation between urban and rural sampling sites. The results indicate that genetic diversity of E. coli populations was not only high in the secondary habitat but also varied with season, flow conditions and, to a lesser extent, sampling location. To our knowledge, this is the first report relating E. coli genotype to stream flow.

Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: the tortoise and the hare III.

Although the chloroplast genome contains many noncoding regions, relatively few have been exploited for interspecific phylogenetic and intraspecific phylogeographic studies. In our recent evaluation of the phylogenetic utility of 21 noncoding chloroplast regions, we found the most widely used noncoding regions are among the least variable, but the more variable regions have rarely been employed. That study led us to conclude that there may be unexplored regions of the chloroplast genome that have even higher relative levels of variability. To explore the potential variability of previously unexplored regions, we compared three pairs of single-copy chloroplast genome sequences in three disparate angiosperm lineages: Atropa vs. Nicotiana (asterids); Lotus vs. Medicago (rosids); and Saccharum vs. Oryza (monocots). These three separate sequence alignments highlighted 13 mutational hotspots that may be more variable than the best regions of our former study. These 13 regions were then selected for a more detailed analysis. Here we show that nine of these newly explored regions (rpl32-trnL((UAG)), trnQ((UUG))-5'rps16, 3'trnV((UAC))-ndhC, ndhF-rpl32, psbD-trnT((GGU)), psbJ-petA, 3'rps16-5'trnK((UUU)), atpI-atpH, and petL-psbE) offer levels of variation better than the best regions identified in our earlier study and are therefore likely to be the best choices for molecular studies at low taxonomic levels.

Amplification of noncoding chloroplast DNA for phylogenetic studies in lycophytes and monilophytes with a comparative example of relative phylogenetic utility from Ophioglossaceae.

Noncoding DNA sequences from numerous regions of the chloroplast genome have provided a significant source of characters for phylogenetic studies in seed plants. In lycophytes and monilophytes (leptosporangiate ferns, eusporangiate ferns, Psilotaceae, and Equisetaceae), on the other hand, relatively few noncoding chloroplast DNA regions have been explored. We screened 30 lycophyte and monilophyte species to determine the potential utility of PCR amplification primers for 18 noncoding chloroplast DNA regions that have previously been used in seed plant studies. Of these primer sets eight appear to be nearly universally capable of amplifying lycophyte and monilophyte DNAs, and an additional six are useful in at least some groups. To further explore the application of noncoding chloroplast DNA, we analyzed the relative phylogenetic utility of five cpDNA regions for resolving relationships in Botrychium s.l. (Ophioglossaceae). Previous studies have evaluated both the gene rbcL and the trnL(UAA)-trnF(GAA) intergenic spacer in this group. To these published data we added sequences of the trnS(GCU)-trnG(UUC) intergenic spacer + the trnG(UUC) intron region, the trnS(GGA)-rpS4 intergenic spacer+rpS4 gene, and the rpL16 intron. Both the trnS(GCU)-trnG(UUC) and rpL16 regions are highly variable in angiosperms and the trnS(GGA)-rpS4 region has been widely used in monilophyte phylogenetic studies. Phylogenetic resolution was equivalent across regions, but the strength of support for the phylogenies varied among regions. Of the five sampled regions the trnS(GCU)-trnG(UUC) spacer+trnG(UUC) intron region provided the strongest support for the inferred phylogeny.

Chloroplast DNA phylogeny and phylogeography of the North American plums (Prunus subgenus Prunus section Prunocerasus, Rosaceae).

The North American plums are a closely related group that are not easily circumscribed, have overlapping morphologies, and are known to hybridize. We previously showed that the North American plums are a closely related, monophyletic group of taxa with little to no cpDNA sequence divergence between taxa. In that study, we came to the unanticipated conclusion that relationships inferred among the taxa contrast sharply with previously defined groups based on morphological characters. Here the aim was to determine if the primary cpDNA haplotypes identified in our earlier study are confined to the taxa in which they were initially observed. The cpDNA rpL16 intron was sequenced for 207 accessions of the 17 North American plum taxa plus Prunus texana. The results show that many taxa contain more than one of the three primary cpDNA haplotypes. Aside from the results found in sect. Prunocerasus, this study has broader implications for phylogenetics in general. The common practice of choosing a single exemplar to represent a taxon can be profoundly misleading in closely related groups. In hindsight, the possibility existed in our earlier study that we could have chosen a different combination of exemplars, which could have resulted in a different inferred phylogeny.

The tortoise and the hare II: relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis.

Chloroplast DNA sequences are a primary source of data for plant molecular systematic studies. A few key papers have provided the molecular systematics community with universal primer pairs for noncoding regions that have dominated the field, namely trnL-trnF and trnK/matK. These two regions have provided adequate information to resolve species relationships in some taxa, but often provide little resolution at low taxonomic levels. To obtain better phylogenetic resolution, sequence data from these regions are often coupled with other sequence data. Choosing an appropriate cpDNA region for phylogenetic investigation is difficult because of the scarcity of information about the tempo of evolutionary rates among different noncoding cpDNA regions. The focus of this investigation was to determine whether there is any predictable rate heterogeneity among 21 noncoding cpDNA regions identified as phylogenetically useful at low levels. To test for rate heterogeneity among the different cpDNA regions, we used three species from each of 10 groups representing eight major phylogenetic lineages of phanerogams. The results of this study clearly show that a survey using as few as three representative taxa can be predictive of the amount of phylogenetic information offered by a cpDNA region and that rate heterogeneity exists among noncoding cpDNA regions.

Addressing the "hardest puzzle in American pomology:" Phylogeny of Prunus sect. Prunocerasus (Rosaceae) based on seven noncoding chloroplast DNA regions.

Prunus subg. Prunus sect. Prunocerasus (Rosaceae) is a North American taxon with 17 commonly recognized taxa. To test the hypothesis of monophyly for the section we sequenced the trnG and rpL16 introns and the trnH-psbA and trnS-trnG intergenic spacers for at least two representatives of each of the five subgenera in Prunus. Additionally we sampled heavily among Prunus subg. Prunus sections Prunus and Armeniaca and Prunus subg. Amygdalus because these groups are putatively most closely related to Prunocerasus. Once monophyly of sect. Prunocerasus was shown we added the sequences of trnL and rpS16 introns and the trnL-trnF spacer in an attempt to increase resolution within the section. The species of sect. Prunocerasus showed an initial split with P. subcordata, the only species from western North America, sister to the rest of the group. The remaining species fell into three primary clades. Within each of the three primary clades there was little phylogenetic resolution. Lastly, we present evidence that P. texana, previously classified in subg. Amygdalus, may be a plum or at least contain a Prunocerasus chloroplast. This is the first phylogenetic hypothesis presented for sect. Prunocerasus, and the clades recovered contrast sharply with previously defined groups based on morphological characters.