Introduction

Agalinis Raf. (Orobanchaceae), the false foxgloves, comprises about 70 species distributed across the Americas, from Canada to Argentina and Chile. About half of its diversity occurs in North America, mainly in the eastern United States, and the other half in South America, where the genus has centers of diversity in the northern Andes and in the highlands of southeastern Brazil. A densely sampled phylogeny based on six plastid and four nuclear regions placed Esterhazya within the South American clade of Agalinis (Latvis et al. 2024), and the genus now includes five additional species (Souza et al. 2025). Biogeographic reconstruction suggests that Agalinis is about 20 million years old and likely originated in eastern North America, followed by migration into South America, with subsequent species radiations in the Andes and Brazil (Pezzi et al. 2026). Migration scenarios through the Andes and/or South American grasslands remain unclear. In addition to recent ‘dispersification’, the parallel rapid radiations in South America are associated with changes in pollinators and life-history strategies (Pezzi et al. 2026). In North America, all species are bee-pollinated, and most are annuals, whereas in South America, Agalinis includes both bee- and hummingbird-pollinated species (Fig. 1), and most are perennials. South American Agalinis therefore offers a unique opportunity to study the genetic basis of rapid trait evolution during an ongoing species radiation, particularly the roles of ancestral polymorphism sorting and hybridization in the origin of hummingbird pollination. Moreover, many of the ~70 described species have highly restricted distributions in both North and South America, and in the United States several are considered imperiled in at least one state. Here, we sequenced and assembled the genomes of 36 Agalinis species and Aureolaria pedicularia using mostly tissue from herbarium specimens. These data will improve our understanding of species relationships and serve as an important genomic resource for the group, supporting species conservation and, together with the chromosome-level reference genome of Agalinis fasciculata (Pezzi and Latvis 2026), studies of genome and trait evolution.

Figure 1
Figure 1.Collection localities of the 37 specimens sequenced in this study. Pink circles indicate bee-pollinated species and red triangles indicate hummingbird-pollinated species of Agalinis. A draft genome was also assembled for Aureolaria pedicularia (green circle), a close relative of Agalinis. Photos of A. angustifolia, A. communis, A. fasciculata, and A. homalantha by Pedro Pezzi; A. kingsii by Maribeth Latvis; and A. scarlatina by Judith Canne-Hilliker.

Material and Methods

We sampled tissue from herbarium specimens of the Missouri Botanical Garden Herbarium (MO), the Philecology Herbarium (BRIT), the Shirley C. Tucker Herbarium at Louisiana State University (LSU), the University of Florida Herbarium (FLAS), and the University of North Carolina at Chapel Hill Herbarium (NCU). For A. angustifolia, we collected fresh leaf tissue from a population in Diamantina, Minas Gerais, Brazil (18°16′31.4″S, 43°41′08.2″W) and dried it in silica gel. We deposited a voucher specimen at the Herbarium of the Universidade Federal de Minas Gerais (BHCB). We extracted DNA using a DNeasy kit (Qiagen, Redwood City, CA) following the manufacturer’s instructions. Paired-end libraries were prepared with a TruSeq kit (Illumina, San Diego, CA) and sequenced on an Illumina HiSeq platform (2 × 150 bp). We trimmed adapters and low-quality regions with Trimmomatic v0.33 (Bolger et al. 2014) and assembled the cleaned reads with SPAdes v3.15.4 (Bankevich et al. 2012), followed by a finishing step with Zanfona (Kieras et al. 2021). We assessed assembly completeness with BUSCO v6.1.0 against the eudicotyledons_odb12 dataset (Tegenfeldt et al. 2025).

Results and Data Availability

The median and mean ages of the sequenced herbarium specimens were 12 and 17 years, respectively (Table 1), with the oldest specimen being A. divaricata, collected in 1970. Sequencing produced a mean of 258,036,581 raw reads per sample, with the lowest being 143,798,190 for A. tarijensis and the highest being 322,938,654 for A. kingsii. The accession codes for the raw reads and the assembled genomes are provided in Table 1. Although DNA was extracted from historical herbarium specimens (except for A. angustifolia), most genomes showed high completeness, with 24 species exceeding 50% (Fig. 2). These results highlight the value of herbaria as genomic resources, especially for rare and hard-to-collect species.

Figure 2
Figure 2.BUSCO completeness scores for the 37 genomes assembled in this study.
Table 1.Species included in this study, with their associated voucher information, collection year, and NCBI accession numbers for raw reads and assembled genomes. Asterisks (*) indicate species whose sequences were assembled from silica-dried leaf tissue. All other species were sampled from herbarium specimens.
Species Voucher Collection Year SRA Accession Genome Accession
Agalinis albida FLAS116623 1972 SRR35318411 GCA_056108385.1
Agalinis angustifolia* BHCB224033 2025 SRR39261250 GCA_062052791.1
Agalinis aspera MO5805413 2004 SRR39261308 GCA_062052793.1
Agalinis brevifolia MO6065669 2006 SRR39261414 GCA_062052789.1
Agalinis communis FLAS284087 2022 SRR35319423 GCA_056108445.1
Agalinis decemloba NCU140243 2013 SRR35920870 GCA_056108405.1
Agalinis densiflora BRIT617674 2019 SRR35921124 GCA_056644815.1
Agalinis divaricata FLAS294906 1970 SRR35316942 GCA_056108425.1
Agalinis fasciculata FLAS291960 2023 SRR35317428 GCA_056108605.1
Agalinis filicaulis LSU117977 2007 SRR35920842 GCA_056523485.1
Agalinis flexicaulis FLAS290370 2010 SRR35921130 GCA_056902695.1
Agalinis gattingeri NCU450759 2018 SRR35921129 GCA_056108485.1
Agalinis georgiana FLAS235345 2002 SRR35318202 GCA_056319905.1
Agalinis harperi LSU220531 2023 SRR35920872 GCA_056523525.1
Agalinis heterophylla LSU220561 2021 SRR35317045 GCA_056592735.1
Agalinis homalantha BRIT617677 2019 SRR35317913 GCA_056319925.1
Agalinis kingsii FLAS277787 2011 SRR35318410 GCA_056108625.1
Agalinis laxa NCU19864 1999 SRR35317928 GCA_056108465.1
Agalinis linifolia NCU452905 2022 SRR35317046 GCA_056108565.1
Agalinis maritima LSU221519 2023 SRR35318150 GCA_056108645.1
Agalinis navasotensis BRIT23261 1992 SRR35318151 GCA_056319705.1
Agalinis neoscotica NCU38058 1990 SRR35318201 GCA_056591835.1
Agalinis obtusifolia NCU452911 2022 SRR35317882 GCA_056319665.1
Agalinis oligophylla BRIT713665 2020 SRR35920873 GCA_056108505.1
Agalinis pennelii MO4037815 1991 SRR39263670 GCA_062057985.1
Agalinis plukenetii FLAS242274 2014 SRR35920845 GCA_056108545.1
Agalinis pulchella FLAS235361 2000 SRR35921125 GCA_056108585.1
Agalinis reflexidens FLAS240887 1984 SRR35319746 GCA_056108525.1
Agalinis scarlatina FLAS294704 1984 SRR35318210 GCA_056319645.1
Agalinis setacea NCU4538800 2021 SRR35942161 GCA_056108685.1
Agalinis skinneriana MO5813713 2004 SRR39263945 GCA_062052788.1
Agalinis stenantha FLAS240889 1984 SRR35319745 GCA_056319945.1
Agalinis strictifolia BRIT486317 2016 SRR35920866 GCA_056108705.1
Agalinis tarijensis MO6565268 2007 SRR39263655 GCA_062057984.1
Agalinis tenuifolia NCU436723 2022 SRR35923989 GCA_056523405.1
Agalinis viridis LSU220075 2022 SRR35921010 GCA_056523325.1
Aureolaria pedicularia NCU454411 2020 SRR35943154 GCA_056523725.1

Acknowledgments

We thank the Missouri Botanical Garden Herbarium (MO), Philecology Herbarium (BRIT), Shirley C. Tucker Herbarium at Louisiana State University (LSU), University of Florida Herbarium (FLAS), and University of North Carolina at Chapel Hill Herbarium (NCU) for access to the specimens sampled. We also thank the Federal University of Minas Gerais Herbarium (BHCB) for allowing us to deposit vouchers of A. angustifolia. Plant material of A. angustifolia was collected under SISBIO/ICMBio license 96764-1 and exported under SisGen registration A2898C1.

Funding

Sequencing was funded by Iridian Genomes (grant #IRGEN_RG_2021-1345). Fieldwork for the collection of A. angustifolia was supported by University of Arkansas start-up funds for M.L.