Nyree J. C. Zerega

Complex origins of breadfruit (Artocarpus altilis, Moraceae): implications for human migrations in Oceania.

Breadfruit (Artocarpus altilis, Moraceae), a traditional starch crop in Oceania, has enjoyed legendary status ever since its role in the infamous mutiny aboard the H.M.S. Bounty in 1789, yet its origins remain unclear. Breadfruits closest relatives are A. camansi and A. mariannensis. DNA fingerprinting data (AFLP, amplified fragment length polymorphisms) from over 200 breadfruit cultivars, 30 A. camansi, and 24 A. mariannensis individuals were used to investigate the relationships among these species. Multivariate analyses and the identification of species-specific AFLP markers indicate at least two origins of breadfruit. Most Melanesian and Polynesian cultivars appear to have arisen over generations of vegetative propagation and selection from A. camansi. In contrast, most Micronesian breadfruit cultivars appear to be the result of hybridization between A. camansi-derived breadfruit and A. mariannensis. Because breadfruit depends on humans for dispersal, the data were compared to theories on the human colonization of Oceania. The results agree with the well-supported theory that humans settled Polynesia via Melanesia. Additionally, a long-distance migration from eastern Melanesia into Micronesia is supported.

USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISMS (AFLP) TO IDENTIFY BLACK COHOSH (ACTAEA RACEMOSA) 1

The rhizome ofActaea racemosa L., commonly called black cohosh, is a popular botanical dietary supplement used to treat female health concerns. The rhizomes used in black cohosh products are often collected from the wild. To ensure quality control, it is imperative that plants be correctly identified. This paper examines the use of the DNA fingerprinting technique, AFLP, as an analytical means of identifyingA. racemosa from three other closely related sympatric species. To this end, 262 AFLP markers were generated, and one unique fingerprint was identified forA. racemosa, whereas two, six, and eight unique fingerprints were identified for the closely related speciesA. pachypoda, A. cordifolia, andA. podocarpa, respectively. Two commercial black cohosh products were also subjected to AFLP analysis and shown to contain onlyA. racemosa. The results of this study suggest that AFLP analysis may offer a useful method for quality control in the botanical dietary supplements industry.ResumenDas Rhizom vonActaea racemosa L., allgemein als ‘black cohosh’ bezeichnet, ist eine beliebte pflanzliche Diätsergänzung, die für weibliche Gesundheitsprobleme benützt wird. Oft sind die in ‘black cohosh’-Produkten verwendeten Rhizome in freier Natur gesammelt. Um Qualitätskontrolle zu sichern, ist es zwingend, die Pflanzen richtig zu identifizieren. Diese Studie überprüft den Gebrauch der DNA-Fingerabdrucktechnik, AFLP, als analytisches Mittel der Identifizierung, umA. racemosa von drei anderen in ihrer Nähe beheimateten und nah verwandten Spezies zuunterscheiden. Zu diesem Zweck wurden 262 AFLP-Fingerabdrücke erzeugt. Für A. racemosa wurde ein einzigartiger Fingerabdruck identifiziert, während für die nah verwandten Spezies A. pachypoda zwei, A. cordifolia sechs, und A. podocarpa acht einzigartige Fingerabdrücke gefunden wurden. Zwei kommerzielle ‘black cohosh’ -Produkte wurden ebenfalls der AFLP-Analyse unterzogen, wobei nur A. racemosa nachgewiesen werden konnte. Die Resultate dieser Studie zeigen, daβ die AFLP-Technik eine nützliche Methode für die Qualitdtskontrolle in der pflanzlichen Diätsergänzungsindustrie bieten kann.

Systematics and Species Limits of Breadfruit (Artocarpus, Moraceae)

Abstract Breadfruit (Artocarpus, Moraceae) is an important staple in Oceania and throughout much of the tropics. Interpretations of species delimitations among breadfruit and its closest relatives have varied from recognition of one to several species. To better understand the systematics and ultimately the origins of breadfruit, we considered evidence from molecular data. Amplified fragment length polymorphism data for 261 individuals of breadfruit, its closest relatives, putative hybrids, and nine outgroup taxa were analyzed using neighbor joining and parsimony analyses. Three species, A. altilis (domesticated breadfruit), A. camansi, and A. mariannensis, are recognized and the existence of hybrids (A. altilis × A. mariannensis) verified. A revised treatment based on the molecular results, as well as morphological and geographical considerations, is presented.

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.

POLLINATION IN THE NEW GUINEA ENDEMIC ANTIAROPSIS DECIPIENS (MORACEAE) IS MEDIATED BY A NEW SPECIES OF THRIPS, THRIPS ANTIAROPSIDIS SP. NOV. (THYSANOPTERA: THRIPIDAE)

Fig pollination is a well‐known example of obligate mutualism involving specialized fig wasps (Hymenoptera, Agaonidae) and Ficus (Moraceae). However, pollination is poorly understood in Castilleae, the recently identified sister group to Ficus. Here we report the first record of thrips pollination in a member of the paleotropical Castilleae. We used phenological measurements, insect trapping, and pollinator exclusion experiments to investigate the mode of pollination in Antiaropsis decipiens, a monotypic dioecious tree of lowland rainforests in New Guinea. We recorded a new species, described here as Thrips antiaropsidis (Thysanoptera, Thripidae), feeding on Antiaropsis pollen, breeding in the staminate inflorescences, and pollinating the carpellate inflorescences. It appears that thrips are lured from staminate to carpellate inflorescences by deceit. We combine these observations with evidence from the Neotropical Castilleae to suggest that thrips pollination may be common in the sister group to figs. We speculate that entomophily in the common ancestor of Ficus and Castilleae predated the origin of the fig pollination mutualism.

Phylogeny and Recircumscription of Artocarpeae (Moraceae) with a Focus on Artocarpus

Abstract Moraceae is a large (∼1,050 species) primarily tropical family with several economically and ecologically important species. While its monophyly has been well supported in recent studies, relationships within the family at the tribal level and below remain unresolved. Delimitation of the tribe Artocarpeae has been particularly difficult. Classifications based on morphology differ from those based on phylogenetic studies, and all treatments include highly heterogeneous assemblages of genera that seem to represent a cross section of the family. We evaluated chloroplast and nuclear DNA sequence data for 60 Moraceae taxa representing all genera that have been included in past treatments of Artocarpeae and also included species from several other Moraceae tribes and closely related families as outgroups. The data were analyzed using maximum parsimony and maximum likelihood methods and indicate that none of the past treatments of Artocarpeae represent a monophyletic lineage. We present the most complete phylogenetic hypothesis for Artocarpeae and the genus Artocarpus to date. Inflorescence evolution and pollination are briefly discussed and the phylogenetic reconstructions are used to inform a revised treatment of Artocarpeae and the Artocarpus subgenera. The following new combinations are proposed: the genus Prainea is reduced to Artocarpus subgenus Prainea , and the series Cauliflori is raised to Artocarpus subgenus Cauliflori .

Development of Microsatellite Loci in Artocarpus altilis (Moraceae) and Cross-Amplification in Congeneric Species

Premise of the study: Microsatellite loci were isolated and characterized from enriched genomic libraries of Artocarpus altilis (breadfruit) and tested in four Artocarpus species and one hybrid. The microsatellite markers provide new tools for further studies in Artocarpus. Methods and Results: A total of 25 microsatellite loci were evaluated across four Artocarpus species and one hybrid. Twenty-one microsatellite loci were evaluated on A. altilis (241), A. camansi (34), A. mariannensis (15), and A. altilis × mariannensis (64) samples. Nine of those loci plus four additional loci were evaluated on A. heterophyllus (jackfruit, 426) samples. All loci are polymorphic for at least one species. The average number of alleles ranges from two to nine within taxa. Conclusions: These microsatellite primers will facilitate further studies on the genetic structure and evolutionary and domestication history of Artocarpus species. They will aid in cultivar identification and establishing germplasm conservation strategies for breadfruit and jackfruit.

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.

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.

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.