A species concept for Phytophthora "taxon Agathis" 1 – causal agent of kauri dieback in New Zealand 1 In memory of Dr Ross E. Beever Landcare Research, Private Bag 92170, Auckland Mail Centre, Auckland 1142, New Zealand 2 BioDiscovery New Zealand Limited, 24 Balfour Street, Parnell *Corresponding author: weirb@landcareresearch.co.nz *BEVAN S. WEIR, ELSA PADERES, DUCKCHUL PARK, NITISH ANAND, DANIEL J. THAN2 AND STANLEY E. BELLGARD INTRODUCTION • Phytophthora “taxon Agathis” (PTA) has been identified (Beever et al. 2009) as a causal agent of kauri (Agathis australis) dieback in northern New Zealand (Figs 1 & 2). • Originally misidentified as the morphologically similar P. heveae (Gadgil 1974), it has now been established that PTA has different oospore morphology to both P. heveae and P. katsurae (Fig. 3) with which it shares a place in Phytophthora ‘ITS Clade 5’ (Cooke et al. 2000). • Preliminary studies using the ITS region of nrDNA had shown small genetic differences between these species (Beever et al. 2009). Figure 2: Kauri dieback symptoms are widely distributed on Agathis australis in northern New Zealand. Figure 1: Kauri dieback symptoms include collar and lower trunk gummosis and advanced canopy decline. P. katsurae – bullate protuberances P. katsurae (x Cocos) - protuberances PTA - slightly raised protuberances P. heave – smooth walled Figure 3: Markedly different ornamentation distinguish comparablesized oogonia and antheridia (35–37 µm) from isolates within Clade 5. AIMS • Review Clade 5 morphospecies (i.e. P. katsurae and P. heveae) known from ‘indigenous’ communities in tropical forests in Australia and Taiwan. • Quantify variation within and between the morphospecies, distinguished largely on the basis of oospore ornamentation. • Based upon morphological and genetic variation, resolve the species’ boundaries within Clade 5. METHODS • Phytophthora isolates were obtained from international culture collections (including type and reference strains where possible) and fresh collections to represent the known diversity of Clade 5. • 15 genomic loci were screened for their utility in resolving species within Clade 5. Eight were highly informative: ITS (the official fungal barcode), NADH and COX1 (mitochondrial genes), and the nuclear genes Ypt-1, L10, TigA, Enolase, and HSP90. • These sequences were concatenated (with different nucleotide substitution models for each partition), and analysed under Bayesian inference (Fig. 6). NBRC 30435 Castanea Japan • Morphological data were collected for culture size at 7 days at 20°C on CMA, MEA, PDA, and V8A media (Fig. 4); and the NBRC 30434 Castanea Japan NBRC 9753 Castanea Japan morphometrics of oogonium and oospores grown in the dark for 10 days at 20°C on V8A (Fig. 5). 0.63 1 NBRC 30433 Castanea Japan 1 CBS 587.85 soil Taiwan REF 1 Oospore width Radial growth rate at 7 days 0.05 0.05 0.05 0.00 0.20 0.10 density density 0.15 0.15 PDA V8A 0.00 15 20 25 30 35 ICMP 18358 Agathis New Zealand PTA ICMP 16948 Cocos Hawaii P. katsurae (x Cocos) DAR 27023 Eucalyptus soil Australia IMI 131093 Theobroma Malaysia 1 CBS 296.29 Hevea Malaysia TYPE 1 0.10 0.00 diameter (µm) Figure 4: Radial growth rate on four media of species and putative species in Phytophthora Clade 5 shows variation between isolates and growth media. 0.05 0.00 10 media 0.10 0.99 PTA PTA MEA 0.15 0.05 1 ICMP 16949 Cocos Hawaii 0.20 0.05 ICMP 18244 Agathis New Zealand 1 0.00 0.15 0.10 0.82 ICMP 16471 Agathis New Zealand 1 0.20 0.15 CMA 0.05 1 P. katsurae (x Cocos) P. katsurae (x Cocos) 0.00 0.20 P. katsurae P. katsurae 0.05 0.10 10 ICMP 17027 Agathis New Zealand 0.20 0.10 20 1 0.00 0.15 radius (mm) 0.10 0.00 0.20 P. heveae P. katsurae P. katsurae (x Cocos) PTA P. heveae 0.15 P. heveae 0.10 30 ICMP 18737 Castanopsis soil Taiwan 0.20 0.15 40 ICMP 16915 soil Taiwan Oogonium width 0.20 P. katsurae P. heveae ICMP 17964 Forest soil China CBS 954.87 Persea Guatemala CBS 124094 P. multivora Australia 20 25 30 35 40 diameter (µm) Figure 5: Density plots show larger oospore and oogonium widths for PTA than other species and putative species in Clade 5. Vertical dashed lines show mean widths. 45 0.008 Figure 6: PTA form a well-supported clade within Phytophthora Clade 5 isolates, sequenced at 8 loci and analysed under Bayesian inference. Posterior probabilities are shown above nodes, scale bar indicates number of expected changes per site. The outgroup branch is shortened by 50% for display. SPECIES CONCEPT FOR PTA Phytophthora ” taxon Agathis” (PTA) is a distinct species within ‘Clade 5’ (Fig. 6). The topologies of each individual gene tree were broadly congruent except for ITS and YptD, where PTA is indistinguishable from P. katsurae (sensu stricto) sequences. PTA appears most closely related to a group of P. katsurae from Cocos in Hawai’i, which probably represent another new species. Genetic variation within the PTA clade is lower than in P. katsurae or P. heveae, possibly indicating a more recent radiation (and introduction to New Zealand), or insufficient geographic sampling. The most obvious morphological difference between members of Clade 5 Phytophthora is the ornamentation of the oogonia (Fig. 3). There are also consistent patterns in the width of the oogonium (including protuberances) and the oospore. PTA has a significantly larger oospore mean width than P. heveae (p < 2.2e-16), P. katsurae (p < 2.2e-16), and P. katsurae (x Cocos) (p < 1.291e-12). CONCLUSIONS Using support from multi-gene sequence analyses and morphometric data we recognise PTA as a distinct species within Clade 5; P. katsurae isolates ex Cocos from Hawai'i should also be recognised as a separate species. These species will be formally described in a forthcoming publication. REFERENCES Beever RE, Waipara NW, Ramsfield TD, Dick MA, Horner IJ 2009. Kauri (Agathis australis) under threat from Phytophthora? In: Goheen EM, Frankl SJ technical coordinators Proceedings, fourth meeting of IUFRO Working Party S07.02.09. General Technical Report PSW-GTR-221. Albany, CA, USDA Forest Service. pp. 74–85. Cooke DEL, Drenth A, Duncan JM, Wagels G, Brasier CM 2000. A molecular phylogeny of Phytophthora and related oomycetes. Fungal Genetics and Biology 30: 17–32. Gadgil PD 1974. Phytophthora heveae a pathogen of kauri. New Zealand Journal of Forestry Science 4(1): 59–63. PTA is a devastating pathogen of kauri trees in New Zealand. Giving this pathogen a formal name and resolving the taxonomy of related species will bring us closer to understanding the origin of the disease. ACKNOWLEDGEMENTS Nicholas Waipara (Principal Biosecurity Advisor, Auckland Council), Peter Buchanan, Shaun Pennycook, Karyn Hoksbergen, Peter Johnston and Chris Winks (Landcare Research, Tamaki). w w w . l a n d c a r e r e s e a r c h . c o . n z