DNA Sequences Help Solve the Mystery of Albizia’s Origin for Better Management Using Biological Control
Falcataria moluccana is the world’s fastest growing tree...but how did it get to Hawai`i?
Falcataria moluccana (“albizia”) was planted within Hawaii’s Forest Reserve system for several years. However this tree is now recognized as an invasive alien that outcompetes native plants, modifies natural ecosystems and imperils the lives and livelihoods of communities across the islands.
In 1917, Joseph Rock deliberately introduced F. moluccana from the Indonesian islands of Borneo and Java. The real source of these trees remains a mystery, since the initial introductions came from outside of the native range of albizia: the Moluccas; New Guinea; the Bismarck Archipelago; and the Solomon Islands. Solving the mystery of albizia’s origin would help us to discover a specialized biological control agent that is safe and effective for Hawaii’s albizia.
We used DNA sequencing techniques to determine the population structure of the trees in Hawai’i and compare them to samples across the native and invaded ranges.
Collaborators from across the Pacific (French Polynesia, Samoa, Cook Islands, Micronesia, and Palau) have contributed leaf samples, while our team collected from sites in Indonesia and Papua New Guinea while surveying for potential insects to use as biological control agents. Over 160 albizia populations have been collected for analysis to date. The results from this study will help us to focus our efforts to find future biological control agents to bring sustainable, safe, long-term management for this giant, highly invasive tree.
Introduction
Falcataria moluccana is the world’s fastest growing tree...but how did it get to Hawai`i?
Falcataria moluccana (“albizia”) was planted within Hawaii’s Forest Reserve system for several years. However this tree is now recognized as an invasive alien that outcompetes native plants, modifies natural ecosystems and imperils the lives and livelihoods of communities across the islands.
In 1917, Joseph Rock deliberately introduced F. moluccana from the Indonesian islands of Borneo and Java. The real source of these trees remains a mystery, since the initial introductions came from outside of the native range of albizia: the Moluccas; New Guinea; the Bismarck Archipelago; and the Solomon Islands. Solving the mystery of albizia’s origin would help us to discover a specialized biological control agent that is safe and effective for Hawaii’s albizia.
We used DNA sequencing techniques to determine the population structure of the trees in Hawai’i and compare them to samples across the native and invaded ranges.
Collaborators from across the Pacific (French Polynesia, Samoa, Cook Islands, Micronesia, and Palau) have contributed leaf samples, while our team collected from sites in Indonesia and Papua New Guinea while surveying for potential insects to use as biological control agents. Over 160 albizia populations have been collected for analysis to date. The results from this study will help us to focus our efforts to find future biological control agents to bring sustainable, safe, long-term management for this giant, highly invasive tree.
Extraction:
- Samples were ground in liquid nitrogen.
- DNA was extracted using Mackeray-Nagel Nucleospin II Plant DNA extraction kit.
Gene region & Primers:
- Ribosomal genes ETS-18s (external transcribed spacer and the 18s rDNA structural gene) were examined with AcR2 forward primer (designed by J.Miller, CSIRO (Ariati, 2006)) and the IGS reverse primer (Baldwind & Markos, 1998).
- 50 µl PCR reaction included Taq Polymerase from New England Biolabs and 1.5 µl DNA.
- The following PCR program was used:
- 95˚C 4 minutes initial denaturing step
- 35 cyclyes: 95˚C – 1 minute denaturing step
- 60˚C – 1 minute annealing step
- 72˚C – 2 minute elongation step
- 72˚C – 7 minute final elongation step
- QIAquick DNA purification kit was used to clean up PCR reaction products.
- Sanger sequencing was done at UC Davis College of Biological Sciences UCDNA Sequencing Facility.
Genetic analysis:
- 77 ETS18s forward direction sequences were used for preliminary analysis of population structure.
- Outgroups included Acacia koa and Albizia chinensis.
- Sequences were edited using Bioedit and aligned with the ClustalW Neighbor Joining algorithm using 10000 bootstrap iterations.
- Neighbor Joining /UPGMA methods were used to create a phylogentic tree using Bioedit accessory packages.
Methods - DNA sequencing
Extraction:
- Samples were ground in liquid nitrogen.
- DNA was extracted using Mackeray-Nagel Nucleospin II Plant DNA extraction kit.
Gene region & Primers:
- Ribosomal genes ETS-18s (external transcribed spacer and the 18s rDNA structural gene) were examined with AcR2 forward primer (designed by J.Miller, CSIRO (Ariati, 2006)) and the IGS reverse primer (Baldwind & Markos, 1998).
- 50 µl PCR reaction included Taq Polymerase from New England Biolabs and 1.5 µl DNA.
- The following PCR program was used:
- 95˚C 4 minutes initial denaturing step
- 35 cyclyes: 95˚C – 1 minute denaturing step
- 60˚C – 1 minute annealing step
- 72˚C – 2 minute elongation step
- 72˚C – 7 minute final elongation step
- QIAquick DNA purification kit was used to clean up PCR reaction products.
- Sanger sequencing was done at UC Davis College of Biological Sciences UCDNA Sequencing Facility.
Genetic analysis:
- 77 ETS18s forward direction sequences were used for preliminary analysis of population structure.
- Outgroups included Acacia koa and Albizia chinensis.
- Sequences were edited using Bioedit and aligned with the ClustalW Neighbor Joining algorithm using 10000 bootstrap iterations.
- Neighbor Joining /UPGMA methods were used to create a phylogentic tree using Bioedit accessory packages.
Albizia from across the Hawaiian Islands were sampled to profile the invasive population.
Wild and planted albizia collected across the native range.
Leaves were collected from over 180 sites: 21 Hawaii & Pacific Island locations and 161 sites in Indonesia, East Timor and Papua New Guinea.
10 individual plants per site were sampled. Leaflets were placed in permeable tea bags and stored in the desiccant silica gel.
Dried leaves were stored in sealed bags with the desiccant until DNA extraction.
Sampling
Albizia from across the Hawaiian Islands were sampled to profile the invasive population.
Wild and planted albizia collected across the native range.
Leaves were collected from over 180 sites: 21 Hawaii & Pacific Island locations and 161 sites in Indonesia, East Timor and Papua New Guinea.
10 individual plants per site were sampled. Leaflets were placed in permeable tea bags and stored in the desiccant silica gel.
Dried leaves were stored in sealed bags with the desiccant until DNA extraction.
Neighbor joining tree with genetic distances shown. Hawaii & Pacific Island populations highlighted.
- This preliminary data includes 74 F. moluccana samples and 3 samples from closely related out-groups.
- There is strong support for the monophyletic relationship within the species Falcataria moluccana.
- The out-group Acacia koa is the closest relative while Albizia chinensis is more distantly related. This finding is supported in recent molecular taxonomy literature.
- Within F. moluccana there are indications of two potential groups of populations.
- Hawaiian and Pacific Island populations are broadly distributed across the phylogenetic tree but are associated with commercial tree plantation populations from Eastern Indonesia.
- The preliminary data from these sequences does not provide the resolution necessary to show where the invasive populations originated.
Results
Neighbor joining tree with genetic distances shown. Hawaii & Pacific Island populations highlighted.
- This preliminary data includes 74 F. moluccana samples and 3 samples from closely related out-groups.
- There is strong support for the monophyletic relationship within the species Falcataria moluccana.
- The out-group Acacia koa is the closest relative while Albizia chinensis is more distantly related. This finding is supported in recent molecular taxonomy literature.
- Within F. moluccana there are indications of two potential groups of populations.
- Hawaiian and Pacific Island populations are broadly distributed across the phylogenetic tree but are associated with commercial tree plantation populations from Eastern Indonesia.
- The preliminary data from these sequences does not provide the resolution necessary to show where the invasive populations originated.
When the tropical storm from hurricane Iselle hit Hawai`i Island in August 2014, high winds caused albizia trees to fall throughout the Puna district causing widespread damage and major disruptions. The call for biological control and other long-term management for these trees reverberated across the State of Hawaii. This study is part of a project to find natural enemies for the biological control agents to reduce the environmental and economic impacts of albizia. Often the most effective insects and diseases for biocontrol are found where the problem plant population originates.
Our preliminary results show that there is a close association of the Hawaii and Pacific Island populations with the commercial plantation trees in Eastern Indonesia - outside the native range. This makes sense as there were deliberate introductions of high yield timber tree stock to the islands for several decades. Unfortunately we do not have the resolution necessary to show exactly where these populations came from within the native range, yet. Future work will attempt to resolve this issue with sequences from additional gene regions.
Albizia blocking road August 2014 Photo by Mike Metcalf (Big Island Now).
[Photo collage at top] Examples of potential biological control agents collected from albizia in the native range (clockwise from top left: shoot tip mining caterpillar (Lepidoptera: Pyralidae), leaf blotch miner (Diptera: Agromyzidae), Falcataria gall rust fungus (Pucciniales: Uromycladium falcatarium), adult moth of shoot tip miner (Lepidoptera: Pyralidae), leaf beetle (Coleoptera: Chrysomelidae), twig mining weevil (Coleoptera: Curculionidae), and Eriophyid mite galls.)
Conclusion
When the tropical storm from hurricane Iselle hit Hawai`i Island in August 2014, high winds caused albizia trees to fall throughout the Puna district causing widespread damage and major disruptions. The call for biological control and other long-term management for these trees reverberated across the State of Hawaii. This study is part of a project to find natural enemies for the biological control agents to reduce the environmental and economic impacts of albizia. Often the most effective insects and diseases for biocontrol are found where the problem plant population originates.
Our preliminary results show that there is a close association of the Hawaii and Pacific Island populations with the commercial plantation trees in Eastern Indonesia - outside the native range. This makes sense as there were deliberate introductions of high yield timber tree stock to the islands for several decades. Unfortunately we do not have the resolution necessary to show exactly where these populations came from within the native range, yet. Future work will attempt to resolve this issue with sequences from additional gene regions.
Albizia blocking road August 2014 Photo by Mike Metcalf (Big Island Now).
[Photo collage at top] Examples of potential biological control agents collected from albizia in the native range (clockwise from top left: shoot tip mining caterpillar (Lepidoptera: Pyralidae), leaf blotch miner (Diptera: Agromyzidae), Falcataria gall rust fungus (Pucciniales: Uromycladium falcatarium), adult moth of shoot tip miner (Lepidoptera: Pyralidae), leaf beetle (Coleoptera: Chrysomelidae), twig mining weevil (Coleoptera: Curculionidae), and Eriophyid mite galls.)
The Mystery remains but new gene sequences promise finer resolution of population structure.
A number of genes have been used for sequencing other plants in the family Fabaceae, although during our initial work only the ETS region provided consistent PCR results for albizia. We plan to explore additional gene regions and troubleshoot this process with new primers, so we can bring higher resolution to this set of data. In this way, we hope to understand the sources of albizia in Hawai’i, and focus our hunt for potential biological control agents. This may be especially important for highly host specific gall forming insects and plant pathogens that are co-evolved with specific albizia populations.
For example, Falcataria gall rust fungus (Uromycladium falcatarium) is one of the most promising of these potential biocontrol agents. This pathogen has a high degree of genetic diversity linked to individual host populations so it is hoped that a safe and effective subspecies/variety can be found on trees near the source of Hawaii's invasive albizia populations.
Uromycladium falcatarium, Falcataria gall rust fungus on albizia saplings near Yogakarta, Indonesia.
Future Prospects
The Mystery remains but new gene sequences promise finer resolution of population structure.
A number of genes have been used for sequencing other plants in the family Fabaceae, although during our initial work only the ETS region provided consistent PCR results for albizia. We plan to explore additional gene regions and troubleshoot this process with new primers, so we can bring higher resolution to this set of data. In this way, we hope to understand the sources of albizia in Hawai’i, and focus our hunt for potential biological control agents. This may be especially important for highly host specific gall forming insects and plant pathogens that are co-evolved with specific albizia populations.
For example, Falcataria gall rust fungus (Uromycladium falcatarium) is one of the most promising of these potential biocontrol agents. This pathogen has a high degree of genetic diversity linked to individual host populations so it is hoped that a safe and effective subspecies/variety can be found on trees near the source of Hawaii's invasive albizia populations.
Uromycladium falcatarium, Falcataria gall rust fungus on albizia saplings near Yogakarta, Indonesia.
