Echoes of ancient introgression punctuate stable genomic lineages in the evolution of figs.

Studies investigating the evolution of flowering plants have long focused on isolating mechanisms such as pollinator specificity. Some recent studies have proposed a role for introgressive hybridization between species, recognizing that isolating processes such as pollinator specialization may not be complete barriers to hybridization. Occasional hybridization may therefore lead to distinct yet reproductively connected lineages. We investigate the balance between introgression and reproductive isolation in a diverse clade using a densely sampled phylogenomic study of fig trees (Ficus, Moraceae). Codiversification with specialized pollinating wasps (Agaonidae) is recognized as a major engine of fig diversity, leading to about 850 species. Nevertheless, some studies have focused on the importance of hybridization in Ficus, highlighting the consequences of pollinator sharing. Here, we employ dense taxon sampling (520 species) throughout Moraceae and 1,751 loci to investigate phylogenetic relationships and the prevalence of introgression among species throughout the history of Ficus. We present a well-resolved phylogenomic backbone for Ficus, providing a solid foundation for an updated classification. Our results paint a picture of phylogenetically stable evolution within lineages punctuated by occasional local introgression events likely mediated by local pollinator sharing, illustrated by clear cases of cytoplasmic introgression that have been nearly drowned out of the nuclear genome through subsequent lineage fidelity. The phylogenetic history of figs thus highlights that while hybridization is an important process in plant evolution, the mere ability of species to hybridize locally does not necessarily translate into ongoing introgression between distant lineages, particularly in the presence of obligate plant-pollinator relationships.

Phylogenomic analyses of the Neotropical Artocarpeae (Moraceae) reveal a history of introgression and support the reinstatement of Acanthinophyllum.

This molecular study of the Neotropical Artocarpeae, the closest living allies of the Asia-Pacific breadfruit genus, uses phylogenomic and network analyses to untangle the evolutionary history of this difficult group. Results paint a picture of a rapid radiation, with introgression, incomplete lineage sorting, and lack of gene tree resolution confounding attempts to reconstruct a well-supported bifurcating tree. While coalescent-based species trees were markedly at odds with morphology, multifurcating phylogenetic network analyses recovered multiple histories, with clearer traces of morphological alliances. The sole unambiguous finding is the sister relationship between Clarisia sect. Acanthinophyllum and the rest of the Neotropical Artocarpeae; as a result, the genus Acanthinophyllum is reinstated.

Paralogs and off-target sequences improve phylogenetic resolution in a densely-sampled study of the breadfruit genus (Artocarpus, Moraceae).

We present a 517-gene phylogenetic framework for the breadfruit genus Artocarpus (ca. 70 spp., Moraceae), making use of silica-dried leaves from recent fieldwork and herbarium specimens (some up to 106 years old) to achieve 96% taxon sampling. We explore issues relating to assembly, paralogous loci, partitions, and analysis method to reconstruct a phylogeny that is robust to variation in data and available tools. While codon partitioning did not result in any substantial topological differences, the inclusion of flanking non-coding sequence in analyses significantly increased the resolution of gene trees. We also found that increasing the size of datasets increased convergence between analysis methods but did not reduce gene tree conflict. We optimized the HybPiper targeted-enrichment sequence assembly pipeline for short sequences derived from degraded DNA extracted from museum specimens. While the subgenera of Artocarpus were monophyletic, revision is required at finer scales, particularly with respect to widespread species. We expect our results to provide a basis for further studies in Artocarpus and provide guidelines for future analyses of datasets based on target enrichment data, particularly those using sequences from both fresh and museum material, counseling careful attention to the potential of off-target sequences to improve resolution.

Land bridges in the Pleistocene contributed to flora assembly on the continental islands of South China: Insights from the evolutionary history of Quercus championii.

The South China Mainland (SCM) and its adjacent continental islands are a global biodiversity hotspot. However, how and when plants dispersed between SCM and Hainan/Taiwan Islands remains largely unknown. In this study, we used restriction site-associated DNA sequencing (RAD-seq) to identify the demographic dynamics and local adaptation of Quercus championii, a dominant forests tree distributed in SCM and Hainan/Taiwan Islands. Through phylogenetic reconstruction, principal components analysis (PCA) and structure analysis, we identified four distinct Q. championii lineages that correspond to its geographical distribution. The genetic structure of Hainan Island population was distinct, possibly reflecting an introgression. We conducted an approximate Bayesian computation analyses and found that Q. championii originated from Southwest China-Northern Vietnam, then dispersed to Southeast China as the climate warmed. During the Pleistocene glacial period, land bridges arose between SCM and Hainan/Taiwan Islands, and the land bridges likely facilitated species dispersal from SCM to these islands. We found a strong correlation between genetic variation and isothermality through a gradient forest analysis and identified precipitation seasonality as a key driver to the local adaptation of Q. championii. Finally, we analyzed putative adaptation loci and identified genes regulating vegetative and reproductive organ development as important for the adaptation of Q. championii to heterogeneous environments. We provide new insights into the evolutionary history and local adaptation of biotas in Southern China and adjacent islands.

Bridging the micro- and macroevolutionary levels in phylogenomics: Hyb-Seq solves relationships from populations to species and above.

Reconstructing phylogenetic relationships at the micro- and macroevoutionary levels within the same tree is problematic because of the need to use different data types and analytical frameworks. We test the power of target enrichment to provide phylogenetic resolution based on DNA sequences from above species to within populations, using a large herbarium sampling and Euphorbia balsamifera (Euphorbiaceae) as a case study. Target enrichment with custom probes was combined with genome skimming (Hyb-Seq) to sequence 431 low-copy nuclear genes and partial plastome DNA. We used supermatrix, multispecies-coalescent approaches, and Bayesian dating to estimate phylogenetic relationships and divergence times. Euphorbia balsamifera, with a disjunct Rand Flora-type distribution at opposite sides of Africa, comprises three well-supported subspecies: western Sahelian sepium is sister to eastern African-southern Arabian adenensis and Macaronesian-southwest Moroccan balsamifera. Lineage divergence times support Late Miocene to Pleistocene diversification and climate-driven vicariance to explain the Rand Flora pattern. We show that probes designed using genomic resources from taxa not directly related to the focal group are effective in providing phylogenetic resolution at deep and shallow evolutionary levels. Low capture efficiency in herbarium samples increased the proportion of missing data but did not bias estimation of phylogenetic relationships or branch lengths.

Allele phasing has minimal impact on phylogenetic reconstruction from targeted nuclear gene sequences in a case study of Artocarpus.

PREMISE OF THE STUDY: Untapped information about allele diversity within populations and individuals (i.e., heterozygosity) could improve phylogenetic resolution and accuracy. Many phylogenetic reconstructions ignore heterozygosity because it is difficult to assemble allele sequences and combine allele data across unlinked loci, and it is unclear how reconstruction methods accommodate variable sequences. We review the common methods of including heterozygosity in phylogenetic studies and present a novel method for assembling allele sequences from target-enriched Illumina sequencing libraries. METHODS: We performed supermatrix phylogeny reconstruction and species tree estimation of Artocarpus based on three methods of accounting for heterozygous sequences: a consensus method based on de novo sequence assembly, the use of ambiguity characters, and a novel method for incorporating read information to phase alleles. We characterize the extent to which highly heterozygous sequences impeded phylogeny reconstruction and determine whether the use of allele sequences improves phylogenetic resolution or decreases topological uncertainty. KEY RESULTS: We show here that it is possible to infer phased alleles from target-enriched Illumina libraries. We find that highly heterozygous sequences do not contribute disproportionately to poor phylogenetic resolution and that the use of allele sequences for phylogeny reconstruction does not have a clear effect on phylogenetic resolution or topological consistency. CONCLUSIONS: We provide a framework for inferring phased alleles from target enrichment data and for assessing the contribution of allelic diversity to phylogenetic reconstruction. In our data set, the impact of allele phasing on phylogeny is minimal compared to the impact of using phylogenetic reconstruction methods that account for gene tree incongruence.

Origin and diversity of an underutilized fruit tree crop, cempedak (Artocarpus integer, Moraceae).

PREMISE OF THE STUDY: Underutilized crops and their wild relatives are important resources for crop improvement and food security. Cempedak [Artocarpus integer (Thunb). Merr.] is a significant crop in Malaysia but underutilized elsewhere. Here we performed molecular characterization of cempedak and its putative wild relative bangkong (Artocarpus integer (Thunb). Merr. var. silvestris Corner) to address questions regarding the origin and diversity of cempedak. METHODS: Using data from 12 microsatellite loci, we assessed the genetic diversity and genetic/geographic structure for 353 cempedak and 175 bangkong accessions from Malaysia and neighboring countries and employed clonal analysis to characterize cempedak cultivars. We conducted haplotype network analyses on the trnH-psbA region in a subset of these samples. We also analyzed key vegetative characters that reportedly differentiate cempedak and bangkong. KEY RESULTS: We show that cempedak and bangkong are sister taxa and distinct genetically and morphologically, but the directionality of domestication origin is unclear. Genetic diversity was generally higher in bangkong than in cempedak. We found a distinct genetic cluster for cempedak from Borneo as compared to cempedak from Peninsular Malaysia. Finally, cempedak cultivars with the same names did not always share the same genetic fingerprint. CONCLUSIONS: Cempedak origins are complex, with likely admixture and hybridization with bangkong, warranting further investigation. We provide a baseline of genetic diversity of cempedak and bangkong in Malaysia and found that germplasm collections in Malaysia represent diverse coverage of the four cempedak genetic clusters detected.

Phylogeny and biogeography of Maclura (Moraceae) and the origin of an anachronistic fruit.

BACKGROUND AND AIMS: Maclura (ca. 12spp., Moraceae) is a widespread genus of trees and woody climbers found on five continents. Maclura pomifera, the Osage orange, is considered a classic example of an anachronistic fruit. Native to the central USA, the grapefruit-sized Osage oranges are unpalatable and have no known extant native dispersers, leading to speculation that the fruits were adapted to extinct megafauna. Our aim was to reconstruct the phylogeny, estimate divergence dates, and infer ancestral ranges of Maclura in order to test the monophyly of subgeneric classifications and to understand evolution and dispersal patterns in this globally distributed group. METHODS: Employing Bayesian and maximum-likelihood methods, we reconstructed the Maclura phylogeny using two nuclear and five chloroplast loci from all Maclura species and outgroups representing all Moraceae tribes. We reconstructed ancestral ranges and syncarp sizes using a family level dated tree, and used Ornstein-Uhlenbeck models to test for significant changes in syncarp size in the Osage orange lineage. KEY RESULTS: Our analyses support a monophyletic Maclura with a Paleocene crown. Subgeneric sections were monophyletic except for the geographically-disjunct Cardiogyne. There was strong support for current species delineations except in the widespread M. cochinchinensis. South America was reconstructed as the ancestral range for Maclura with subsequent colonization of Africa and the northern hemisphere. The clade containing M. pomifera likely diverged in the Oligocene, closely coinciding with crown divergence dates of the mammoth/mastodon and sloth clades that contain possible extinct dispersers. The best fitting model for syncarp size evolution indicated an increase in both syncarp size and the rate of syncarp size evolution in the Osage orange lineage. CONCLUSIONS: We conclude that our findings are consistent with the hypothesis that M. pomifera was adapted to dispersal by extinct megafauna. In addition, we consider dispersal rather than vicariance to be most likely responsible for the present distribution of Maclura, as crown divergence post-dated the separation of Africa and South America. We propose revised sectional delimitations based on the phylogeny. This study represents a complete phylogenetic and biogeographic analysis of this globally distributed genus and provides a basis for future work, including a taxonomic revision.

Out of Borneo: biogeography, phylogeny and divergence date estimates of Artocarpus (Moraceae).

Background and Aims: The breadfruit genus ( Artocarpus , Moraceae) includes valuable underutilized fruit tree crops with a centre of diversity in Southeast Asia. It belongs to the monophyletic tribe Artocarpeae, whose only other members include two small neotropical genera. This study aimed to reconstruct the phylogeny, estimate divergence dates and infer ancestral ranges of Artocarpeae, especially Artocarpus , to better understand spatial and temporal evolutionary relationships and dispersal patterns in a geologically complex region. Methods: To investigate the phylogeny and biogeography of Artocarpeae, this study used Bayesian and maximum likelihood approaches to analyze DNA sequences from six plastid and two nuclear regions from 75% of Artocarpus species, both neotropical Artocarpeae genera, and members of all other Moraceae tribes. Six fossil-based calibrations within the Moraceae family were used to infer divergence times. Ancestral areas and estimated dispersal events were also inferred. Key Results: Artocarpeae, Artocarpus and four monophyletic Artocarpus subgenera were well supported. A late Cretaceous origin of the Artocarpeae tribe in the Americas is inferred, followed by Eocene radiation of Artocarpus in Asia, with the greatest diversification occurring during the Miocene. Borneo is reconstructed as the ancestral range of Artocarpus , with dozens of independent in situ diversification events inferred there, as well as dispersal events to other regions of Southeast Asia. Dispersal pathways of Artocarpus and its ancestors are proposed. Conclusions: Borneo was central in the diversification of the genus Artocarpus and probably served as the centre from which species dispersed and diversified in several directions. The greatest amount of diversification is inferred to have occurred during the Miocene, when sea levels fluctuated and land connections frequently existed between Borneo, mainland Asia, Sumatra and Java. Many species found in these areas have extant overlapping ranges, suggesting that sympatric speciation may have occurred. By contrast, Artocarpus diversity east of Borneo (where many of the islands have no historical connections to the landmasses of the Sunda and Sahul shelves) is unique and probably the product of over water long-distance dispersal events and subsequent diversification in allopatry. This work represents the most comprehensive Artocarpus phylogeny and biogeography study to date and supports Borneo as an evolutionary biodiversity hotspot.

Corrigendum: Phylogenomics of the Andean tetraploid clade of the American Amaryllidaceae (subfamily Amaryllidoideae): Unlocking a polyploid generic radiation abetted by continental geodynamics.

[This corrects the article DOI: 10.3389/fpls.2020.582422.].

Linking breadfruit cultivar names across the globe connects histories after 230 years of separation.

Every crop has a story. The story of breadfruit (Artocarpus altilis), an increasingly valued staple crop in tropical agroforestry systems, is filled with intrigue, oppression, and remains incomplete. The Caribbean is a major producer and consumer of breadfruit, yet most breadfruit there came from a single 1793 introduction aimed at providing a cheap food source for slaves forced to work on British plantations. St. Vincent was the first significant point of Caribbean introduction and played a vital role in subsequent breadfruit distribution throughout the region. Hundreds of cultivars are documented in breadfruit's native Oceania. It remains a mystery, however, which ones were introduced to the Caribbean 230 years ago-still comprising the vast diversity found there today. Integrating local knowledge, historical documents and specimens, morphological data, and DNA, we identify eight major global breadfruit lineages-five of which are found in the Caribbean and likely represent the original 1793 introduction. Genetic data were able to match two Caribbean cultivar names confidently to their Oceania counterparts. Genetics and morphology together enabled additional possible matches. Many other named cultivars within lineages are too genetically similar to differentiate, highlighting difficulties of defining and identifying variation among clonally propagated triploid crops. Breadfruit is important in resilient agroforestry in tropical islands predicted to be especially affected by climate change. Findings reveal global links, building upon collective knowledge that can be used to inform breadfruit management. Results are also summarized in a brochure about breadfruit history and diversity in St. Vincent, and the Caribbean more broadly.

Amis Pacilo and Yami Cipoho are not the same as the Pacific breadfruit starch crop-Target enrichment phylogenomics of a long-misidentified Artocarpus species sheds light on the northward Austronesian migration from the Philippines to Taiwan.

'Breadfruit' is a common tree species in Taiwan. In the indigenous Austronesian Amis culture of eastern Taiwan, 'breadfruit' is known as Pacilo, and its fruits are consumed as food. On Lanyu (Botel Tobago) where the indigenous Yami people live, 'breadfruit' is called Cipoho and used for constructing houses and plank-boats. Elsewhere in Taiwan, 'breadfruit' is also a common ornamental tree. As an essential component of traditional Yami culture, Cipoho has long been assumed to have been transported from the Batanes Island of the Philippines to Lanyu. As such, it represents a commensal species that potentially can be used to test the hypothesis of the northward Austronesian migration 'into' Taiwan. However, recent phylogenomic studies using target enrichment show that Taiwanese 'breadfruit' might not be the same as the Pacific breadfruit (Artocarpus altilis), which was domesticated in Oceania and widely cultivated throughout the tropics. To resolve persistent misidentification of this culturally and economically important tree species of Taiwan, we sampled 36 trees of Taiwanese Artocarpus and used the Moraceae probe set to enrich 529 nuclear genes. Along with 28 archived Artocarpus sequence datasets (representing a dozen taxa from all subgenera), phylogenomic analyses showed that all Taiwanese 'breadfruit' samples, together with a cultivated ornamental tree from Hawaii, form a fully supported clade within the A. treculianus complex, which is composed only of endemic Philippine species. Morphologically, the Taiwanese 'breadfruit' matches the characters of A. treculianus. Within the Taiwanese samples of A. treculianus, Amis samples form a fully supported clade derived from within the paraphyletic grade composed of Yami samples, suggesting a Lanyu origin. Results of our target enrichment phylogenomics are consistent with the scenario that Cipoho was transported northward from the Philippines to Lanyu by Yami ancestors, though the possibility that A. treculianus is native to Lanyu cannot be ruled out completely.

Engagement with indigenous people preserves local knowledge and biodiversity alike.

Indigenous peoples are important stewards of biodiversity, often living near and possessing intimate knowledge of ecosystems. As a result, species new to science may be long known to indigenous people. While the scientific endeavor has long benefitted from indigenous knowledge, it has usually not engaged with it on equal footing1,2. While Linnaean taxonomy offers a broad framework for global comparisons, it may lack the detailed local insights possessed by indigenous peoples. This study illustrates how meaningful engagement with indigenous knowledge - throughout the scientific process - can improve biodiversity science and promote conservation1,2, particularly in studies of crop wild relatives, an international priority3 for food security in the face of climate change4. Two species of fruit trees recognized as distinct by the Iban and Dusun peoples, but considered a single species in current Linnaean taxonomy, were confirmed as distinct taxa by molecular studies. They correspond to Artocarpus odoratissimus Blanco and Artocarpus mutabilis Becc., whose distinguishing characteristics were clarified by members of indigenous communities.

Plantago campestris (Plantaginaceae), a rare new species from southern Brazil, supported by phylogenomic and morphological evidence.

High-throughput sequencing, when combined with taxonomic expertise, is a powerful tool to refine and advance taxonomic classification, including at the species level. In the present work, a new species, Plantago campestris, is described out of the P. commersoniana species complex, based on phylogenomic and morphological evidence. The main morphological characters that distinguish the new species from P. commersoniana are the glabrous posterior sepals and the slightly broader leaves. The new species is known from only three localities, all in natural high-elevation grasslands in Paraná and Santa Catarina states, southern Brazil. According to the IUCN criteria new species should be assessed as Endangered (EN). We present field photographs of P. campestris and related species, and we provide an identification key to the species previously included within the circumscription of P. commersoniana.

Phylogenomics of the Andean Tetraploid Clade of the American Amaryllidaceae (Subfamily Amaryllidoideae): Unlocking a Polyploid Generic Radiation Abetted by Continental Geodynamics.

One of the two major clades of the endemic American Amaryllidaceae subfam. Amaryllidoideae constitutes the tetraploid-derived (n = 23) Andean-centered tribes, most of which have 46 chromosomes. Despite progress in resolving phylogenetic relationships of the group with plastid and nrDNA, certain subclades were poorly resolved or weakly supported in those previous studies. Sequence capture using anchored hybrid enrichment was employed across 95 species of the clade along with five outgroups and generated sequences of 524 nuclear genes and a partial plastome. Maximum likelihood phylogenetic analyses were conducted on concatenated supermatrices, and coalescent-based species tree analyses were run on the gene trees, followed by hybridization network, age diversification and biogeographic analyses. The four tribes Clinantheae, Eucharideae, Eustephieae, and Hymenocallideae (sister to Clinantheae) are resolved in all analyses with > 90 and mostly 100% support, as are almost all genera within them. Nuclear gene supermatrix and species tree results were largely in concordance; however, some instances of cytonuclear discordance were evident. Hybridization network analysis identified significant reticulation in Clinanthus, Hymenocallis, Stenomesson and the subclade of Eucharideae comprising Eucharis, Caliphruria, and Urceolina. Our data support a previous treatment of the latter as a single genus, Urceolina, with the addition of Eucrosia dodsonii. Biogeographic analysis and penalized likelihood age estimation suggests an origin in the Cauca, Desert and Puna Neotropical bioprovinces for the complex in the mid-Oligocene, with more dispersals than vicariances in its history, but no extinctions. Hymenocallis represents the only instance of long-distance vicariance from the tropical Andean origin of its tribe Hymenocallideae. The absence of extinctions correlates with the lack of diversification rate shifts within the clade. The Eucharideae experienced a sudden lineage radiation ca. 10 Mya. We tie much of the divergences in the Andean-centered lineages to the rise of the Andes, and suggest that the Amotape-Huancabamba Zone functioned as both a corridor (dispersal) and a barrier to migration (vicariance). Several taxonomic changes are made. This is the largest DNA sequence data set to be applied within Amaryllidaceae to date.

Strategies for reducing per-sample costs in target capture sequencing for phylogenomics and population genomics in plants.

The reduced cost of high-throughput sequencing and the development of gene sets with wide phylogenetic applicability has led to the rise of sequence capture methods as a plausible platform for both phylogenomics and population genomics in plants. An important consideration in large targeted sequencing projects is the per-sample cost, which can be inflated when using off-the-shelf kits or reagents not purchased in bulk. Here, we discuss methods to reduce per-sample costs in high-throughput targeted sequencing projects. We review the minimal equipment and consumable requirements for targeted sequencing while comparing several alternatives to reduce bulk costs in DNA extraction, library preparation, target enrichment, and sequencing. We consider how each of the workflow alterations may be affected by DNA quality (e.g., fresh vs. herbarium tissue), genome size, and the phylogenetic scale of the project. We provide a cost calculator for researchers considering targeted sequencing to use when designing projects, and identify challenges for future development of low-cost sequencing in non-model plant systems.

Low-coverage, whole-genome sequencing of Artocarpus camansi (Moraceae) for phylogenetic marker development and gene discovery.

PREMISE OF THE STUDY: We used moderately low-coverage (17×) whole-genome sequencing of Artocarpus camansi (Moraceae) to develop genomic resources for Artocarpus and Moraceae. METHODS AND RESULTS: A de novo assembly of Illumina short reads (251,378,536 pairs, 2 × 100 bp) accounted for 93% of the predicted genome size. Predicted coding regions were used in a three-way orthology search with published genomes of Morus notabilis and Cannabis sativa. Phylogenetic markers for Moraceae were developed from 333 inferred single-copy exons. Ninety-eight putative MADS-box genes were identified. Analysis of all predicted coding regions resulted in preliminary annotation of 49,089 genes. An analysis of synonymous substitutions for pairs of orthologs (Ks analysis) in M. notabilis and A. camansi strongly suggested a lineage-specific whole-genome duplication in Artocarpus. CONCLUSIONS: This study substantially increases the genomic resources available for Artocarpus and Moraceae and demonstrates the value of low-coverage de novo assemblies for nonmodel organisms with moderately large genomes.

HybPiper: Extracting coding sequence and introns for phylogenetics from high-throughput sequencing reads using target enrichment.

PREMISE OF THE STUDY: Using sequence data generated via target enrichment for phylogenetics requires reassembly of high-throughput sequence reads into loci, presenting a number of bioinformatics challenges. We developed HybPiper as a user-friendly platform for assembly of gene regions, extraction of exon and intron sequences, and identification of paralogous gene copies. We test HybPiper using baits designed to target 333 phylogenetic markers and 125 genes of functional significance in Artocarpus (Moraceae). METHODS AND RESULTS: HybPiper implements parallel execution of sequence assembly in three phases: read mapping, contig assembly, and target sequence extraction. The pipeline was able to recover nearly complete gene sequences for all genes in 22 species of Artocarpus. HybPiper also recovered more than 500 bp of nontargeted intron sequence in over half of the phylogenetic markers and identified paralogous gene copies in Artocarpus. CONCLUSIONS: HybPiper was designed for Linux and Mac OS X and is freely available at https://github.com/mossmatters/HybPiper.

Chloroplast microsatellite markers for Artocarpus (Moraceae) developed from transcriptome sequences.

PREMISE OF THE STUDY: Chloroplast microsatellite loci were characterized from transcriptomes of Artocarpus altilis (breadfruit) and A. camansi (breadnut). They were tested in A. odoratissimus (terap) and A. altilis and evaluated in silico for two congeners. METHODS AND RESULTS: Fifteen simple sequence repeats (SSRs) were identified in chloroplast sequences from four Artocarpus transcriptome assemblies. The markers were evaluated using capillary electrophoresis in A. odoratissimus (105 accessions) and A. altilis (73). They were also evaluated in silico in A. altilis (10), A. camansi (6), and A. altilis × A. mariannensis (7) transcriptomes. All loci were polymorphic in at least one species, with all 15 polymorphic in A. camansi. Per species, average alleles per locus ranged between 2.2 and 2.5. Three loci had evidence of fragment-length homoplasy. CONCLUSIONS: These markers will complement existing nuclear markers by enabling confident identification of maternal and clone lines, which are often important in vegetatively propagated crops such as breadfruit.