Next-generation sequencing reveals cryptic mtDNA diversity of Plasmodium relictum in the Hawaiian Islands S. I. JARVI 1 *, M. E. FARIAS 1 , D. A. LAPOINTE 2 , M. BELCAID 3 and C. T. ATKINSON 2 1 Department of Pharmaceutical Sciences, Daniel K. Inouye College of Pharmacy, University of Hawaii at Hilo, 34 Rainbow Drive, Hilo, HI 96720, USA 2 U.S. Geological Survey, Pacific Island Ecosystems Research Center, Kilauea Field Station, P.O. Box 44, Building 343, Hawaii National Park, HI 96718, USA 3 Information and Computer Sciences Department, University of Hawaii at Manoa, Pacific Ocean Science and Technology Building, Room 317, 1680 East-West Road, Honolulu, HI 96822, USA (Received 10 January 2013; revised 12 April and 22 May 2013; accepted 23 May 2013; first published online 19 August 2013) SUMMARY Next-generation 454 sequencing techniques were used to re-examine diversity of mitochondrial cytochrome b lineages of avian malaria (Plasmodium relictum) in Hawaii. We document a minimum of 23 variant lineages of the parasite based on single nucleotide transitional changes, in addition to the previously reported single lineage (GRW4). A new, publicly available portal (Integroomer) was developed for initial parsing of 454 datasets. Mean variant prevalence and frequency was higher in low elevation Hawaii Amakihi (Hemignathus virens) with Avipoxvirus-like lesions (P = 0·001), suggesting that the variants may be biologically distinct. By contrast, variant prevalence and frequency did not differ significantly among mid-elevation Apapane (Himatione sanguinea) with or without lesions (P = 0·691). The low frequency and the lack of detection of variants independent of GRW4 suggest that multiple independent introductions of P. relictum to Hawaii are unlikely. Multiple variants may have been introduced in heteroplasmy with GRW4 or exist within the tandem repeat structure of the mitochondrial genome. The discovery of multiple mitochondrial lineages of P. relictum in Hawaii provides a measure of genetic diversity within a geographically isolated population of this parasite and suggests the origins and evolution of parasite diversity may be more complicated than previously recognized. Key words: mtDNA, avian malaria, Hawaii, Plasmodium relictum, next generation sequencing, Integroomer. INTRODUCTION Ecological studies of avian haemosporidian parasites based largely on PCR amplification and direct sequencing of short segments of parasite mitochondrial genes have grown exponentially in number over the past decade (Bensch et al. 2009). Single nucleotide substitutions have been used to identify unique lineages that are often used as discrete taxa for studies of geographical distribution, host specificity and parasite–vector associations. These studies have revealed an unprecedented amount of diversity among these organisms that is often difficult to reconcile with more traditional species concepts based on morphological and biological character- istics. As a rule of thumb, haemosporidians that differ by more than 5% in mitochondrial sequence appear to be distinguishable by morphological features of erythrocytic stages of the parasites (Hellgren et al. 2007; Valkiūnas et al. 2009), with most diversity captured by sequencing a 479 bp region of the cytochrome b gene (Hellgren et al. 2007). In reality though, we know little about how to define these parasites as biological species because so little is known about their basic life cycles, vectors and epizootiology. This problem is illustrated by the remarkable diversity of mtDNA lineages that have been associated with Plasmodium relictum, one of the most widely distributed species of avian Plasmodium both in terms of host range and geographical distribution (Beadell et al. 2006). In an attempt to place isolates of P. relictum from the Hawaiian Islands into a global phylogenetic context, Beadell et al. (2006) identified 31 distinct lineages from around the world in two well- supported clades which included lineages identified as P. relictum by established morphological criteria. A single lineage of Plasmodium (lineage 15, previously identified as lineage GRW4, Bensch et al. 2000) was identified in 75 infected introduced and endemic forest birds from Hawaii, Maui, Oahu and Kauai. The authors concluded that a single lineage of the parasite had been introduced to the islands and that the recent expansion of native forest bird populations in some low-elevation habitats (Woodworth et al. 2005) had likely not been facilitated by cryptic introduction of different parasite lineages of lower virulence. However, their report relied on results of traditional direct sequencing, which fails to detect related lineages making up less than 5–25% * Corresponding author. Department of Pharmaceutical Sciences, Daniel K. Inouye College of Pharmacy, University of Hawaii at Hilo, 34 Rainbow Drive, Hilo, HI 96720, USA. E-mail: jarvi@hawaii.edu 1741 Parasitology (2013), 140, 1741–1750. © Cambridge University Press 2013 doi:10.1017/S0031182013000905