Ignition Grant Round 6 (July 2016)

Bee Gunn (ANU) and Sarah Mathews (CSIRO)

Introduction

Palms are globally important angiosperms in the tropics and subtropics. They are second only to grasses in economic importance, and include oil, date, and coconut palms. Their unique structural architecture and their high diversity and abundance make them compelling to morphologists, tropical biologists, horticulturalists, and the public.

Despite their allure, their unusual morphology, woodiness, and remote tropical habitats make them challenging to study. Australia offers unique opportunities for insights into palm diversity. Members of all five subfamilies of the palm family (Arecaceae) occur here, distributed in 10 genera and comprising lineages that range from being very narrowly to more broadly distributed. Endemicity is high in the monsoonal wet tropics and savanna forests in northern Australia, Lord Howe Island, and the arid centre.

Palms were a significant component of mesic forests that occurred across Australia in the Oligocene/ Miocene (ca. 23 Mya), and their present day ranges and high levels of endemicity suggest that at least some current palm distributions are relictual. Some, however, may result from more recent diversification events after migration from neighboring continents, as is the case for Livistona.

Palm fruits are important to native fauna, which often serve as seed dispersers. In particular, fleshy-fruited palms such as Normanbya normanbyi, Archontophoenix cunninghamiana, and Livistona australis provide food for frugivores. Normanbya (black palm) is monotypic and endemic to Cape Tribulation, where its fruits are consumed by the endangered southern cassowary, white tailed rats, musky rat-kangaroo, and possibly bats, especially during the dry season. The dispersed fruits play a role in rainforest regeneration, and the phenology of Normanbya suggests that it is a key provider of nectar and pollen for insects during the dry season. Palms are therefore an excellent system for coevolutioanry studies, which would in turn provide data bearing on ecological resilience in their habitats.

The use of palms by indigenous Australians has a long history. Livistona mariae, an endemic in central Australia, was likely introduced into that region by Australian Aboriginals around 30,000 years ago for use as food and medicine. The cabbage (heart) of another species, L. humilis, were roasted for consumption. Normanbya is significant to the Daintree kuku yalanji people because the hard wood makes good spears and the palm heart is used for making bags.

Aims

The overarching goal of our study is to develop an array of hyRAD probes for hybridisation-based sequence capture to enable genome-scale approaches and the use of herbarium specimens in studies of palm evolution and diversity. An early-stage target, achievable during the ignition grant phase, is the resolution of phylogenetic relationships in subtribe Ptychospermatinae.

Ptychospermatinae includes five endemics from northern Australia belonging to a clade with lineages from Southeast Asia, New Guinea, and the western Pacific:

• Normanbya (1 sp, endemic to northern Queensland)
• Drymophloeus (8 sp: New Guinea, Moluccas, Bismarck Archipelago, Solomon Islands, and Samoa)
• Carpentaria (1 sp, endemic to N.T.), Wodyetia (1 sp, endemic to north-east Queensland)
• Ptychosperma (31 sp: Moluccas, New Guinea, and Australia, with two Australian endemics)
• Ptychococcus (2 sp: New Guinea, Bismarck Archipelago, and Bougainville)
• Balaka (11 sp: Fiji and Samoa)
• Solfia (1 sp: Samoa)
• Ponapea (3 sp: Caroline Islands)
• Bassiophoenix (2 sp: New Guinea)
• Adonidia (1 sp: Northern Borneo)
• Veitchia (8 sp: Vanuatu, Fiji, and Tonga)

Methods and Materials 

Probe design We propose to develop hyRAD protocols to enrich shotgun libraries of Ptychospermatinae. First, high quality DNA extracted from fresh samples will be double digested (ddRAD) by two restriction enzymes  (RE). The resulting ddRAD fragments cut by the two RE will be size selected using the LabChip GXII. Fragments in the ddRAD library are then used as hybridization-capture probes to enrich shotgun libraries from either herbarium or fresh material. The workflow will be:

1. generate ddRAD library from high quality DNA samples
2. biotinylate for the next part of the protocol
3. prepare libraries
4. hybridization capture and barcoding of fragments
5. sequence the enriched shotgun libraries and the ddRAD library (optional) on Illumina HiSeq 2500 platform
6. treatment of hyRAD sequences using bioinformatics pipeline for contig assembly, alignment to ddRAD library and SNP calling.

Target taxa Sub-tribal sampling: Ptychospermatinae - our target genera are Normanbya, Drymophloeus, Carpentaria and Wodyetia, Ptychosperma, Ptychococcus, Balaka, Solfia, Ponopea, Bassiophoenix, Adonidia and Veitchia. We will obtain fresh leaf samples from the living palm collections at Royal Botanic Gardens, Sydney for generating the ddRAD probes. To test the utility of the probes at the family level - we will sample Australian palm genera representing each of the five subfamilies: Calamoideae (Calamus); Nypoideae (Nypa); Coryphoideae (Livistona), Ceroxyloideae (Oraniopsis) and Arecoideae (Archonotophoenix, Cocos, Howea, Linospadix). 

Use of herbarium materials The Australian National Herbarium holds collections of palm specimens from northern Australia and is the largest repository of plants from Papua New Guinea outside PNG.  We will exploit these historical collections and silica-dried leaf material to provide genomic DNA for the shotgun libraries. We aim to sample about 70% or more of the Ptychospermatinae, depending on the availability of samples from fresh or herbarium collections.

Significance of study

This will resolve the phylogeny of Australian Ptychospermatinae, which are of conservation concern. Land fragmentation has threatened endemic palm species in this clade, which are associated with important food resources for Australian fauna. This interaction is highlighted in the coevolutionary relationship between the black palm (Normanbya normanbyi) and southern cassowary, which are both listed as threatened and endangered respectively under the IUCN Red List. Another rare palm growing in the Northern Territory, Ptychosperma blesseri is under threat due to hybridization with introduced Ptychosperma species.

Future directions

These hybridization probes will enable us to address wider questions about Australian palm diversity and evolution in future studies. For example:

1. A dated phylogeny of all Australian palm genera from genomic scale data would help us understand patterns of palm diversifications in Australia. We hypothesize that lineages of Livistona responded to aridification and were able to occupy niches in the moist ravines across Australia, whereas the species poor lineages, such as most Ptychospermatinae, were confined to moist rainforests that shrunk during the Miocene.
2. Population-level sampling of relevant lineages would help us understand the evolutionary dynamics or drivers of palm diversity across Northern Australia, Papua New Guinea, Pacific and South East Asia.
3. Interactions between Australian endemic palms and endemic fauna can also be examined in the context of genetic and phylogenetic data from palms, combined with data on climate, animal interactions, and fruit types.