Species Limits in Avian Malaria Parasites (Haemosporida): How to Move Forward in the Molecular Era

1223 REVIEW ARTICLE

Species limits in avian malaria parasites (Haemosporida): how to move forward in the molecular era

DIANA C. OUTLAW1* and ROBERT E. RICKLEFS2 1 Department of Biological Sciences, Mississippi State University, Mississippi State, MS 39759, USA 2 Department of Biology, University of Missouri-St. Louis, One University Boulevard, St. Louis, MO 63121, USA

(Received 4 November 2013; revised 20 December 2013, 3 March and 7 March 2014; accepted 13 March 2014; first published online 9 May 2014)

SUMMARY

Delimiting species of malaria parasites (Haemosporida) has become increasingly problematic as new lineages of parasites are identified solely by molecular information, particularly mitochondrial cytochrome b sequence data. In this review, we highlight some of the issues, both historical and contemporary, that have hindered the development of objective criteria to diagnose, delimit and define species of haemosporidians. Defining species is not the focal interest of most researchers, most of whom merely wish to determine whether lineages identified in their samples match those of other researchers, and if so, where and in which host species. Rather than revisiting all the issues with respect to delimiting and naming species, we instead focus on finding a practical near-term resolution to the ‘species problem’ that utilizes the community’s largest resource: mitochondrial cytochrome b DNA sequences. We recommend a standardized procedure to ‘tag’ these sequences, based on per cent sequence similarity, that will allow researchers to directly assess the novelty, known hosts and geographic distribution of avian malaria parasite lineages.

Key words: Avian malaria, species limits, Haemosporida.

THE SPECIES PROBLEM: WHY NOW? parasites has increased rapidly since 1996 (Scopus ‘avian malaria, cytochrome b’ search by year; To understand evolutionary processes that have Figure S1), and more than 1000 mitochondrial generated the biodiversity of haemosporidian mala- cytochrome b (cyt b) sequences have been recog- ria parasites, we need to circumscribe units of bio- nized among avian malaria parasites (*1500 as diversity – species or, at least, operational taxonomic of February, 2014; MalAvi database; Bensch et al. units (OTUs). Recent discussion of the evolutionary 2009). Although disagreement continues about the history and diversification of these parasites has been relevance of sequence differences to species bound- encumbered by disagreement over metrics that can aries in protists and bacteria (Adl et al. 2007; (or should) be used to diagnose and delimit species. Morrison, 2009; Pérez-Ponce de Léon and Nadler, Although human malaria parasite species are well- 2010), molecular studies of parasite diversity con- defined and well-studied, hundreds of morphologi- tinue at an accelerating pace. As genomic data become cally described species of malaria parasites infect increasingly available, it is timely to discuss the ap- birds (*200), lizards (*150) and non-anthropoid plication of molecular tools to defining species limits. mammals (*20 known), and an increasing number Certainly, delimiting species is prerequisite to under- of genetic lineages (i.e. cytochrome b haplotypes) standing diversity, distribution and speciation in have yet to be attributed to formally described avian malaria parasites. species. Molecular studies over the last 20 years, Formal descriptions of haemosporidian parasites, beginning with Escalante and Ayala (1994), have with the exception of Plasmodium, are based pri- revealed a previously unexpected level of diversity in marily on limited morphological characters observed these parasites, particularly those of birds, that rivals in the mature gametocytes preserved on blood smears that of their host species (e.g. Bensch et al. 2000, (e.g. Perkins, 2000; Valkiūnas et al. 2009a, b; 2004; Ricklefs and Fallon, 2002; Ricklefs et al. 2004; Paperna and Yosef, 2010; reviewed in Valkiūnas, Beadell et al. 2006). Indeed, the number of papers 2005). Indeed, over the history of haemosporidian per year that identify genetic lineages of avian malaria taxonomy, opinions have differed concerning the * Corresponding author: Department of Biological utility of formally describing a malaria parasite, par- Sciences, Mississippi State University, Mississippi State, ticularly when its species status is uncertain. Alterna- MS 39759, USA. E-mail: [email protected] tively, one could provide an ambiguous designation,

Parasitology (2014), 141, 1223–1232. © Cambridge University Press 2014 doi:10.1017/S0031182014000560

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e.g. Plasmodium bird.1, which might relegate the In a recent study of lizard parasites, Falk et al. (2011) entity to anonymity and minimize its potential found four new species of haemosporidian that contribution to our understanding of malaria parasite were concordant across multi-locus phylogenetic biodiversity (see Garnham, 1966). It is clear from the data, but for which morphological characters were amount of molecular data that has accumulated over overlapping and somewhat ambiguous. Finally, in a the last 10 years, and from our ambiguity about cir- very striking recent example, Merino et al. (2012) cumscribing species, or even OTUs, that we remain describe a new avian parasite species that is geneti- in uncharted territory with regard to taxonomically cally identical in cyt b sequence to another species but informative characters for delimiting species (re- morphologically distinct within one host species viewed in Perkins et al. 2011). An examination of the (see ‘Historical philosophies and approaches’ below literature shows that several new species of malaria for a possible explanation). parasite are described every year and that the criteria used to define them can differ markedly. In some AN OVERVIEW OF AVIAN HAEMOSPORIDIAN cases, species limits are established solely by mol- DIVERSITY ecular data (Rich et al. 2009; Merino et al. 2012; see also Valkiūnas et al. 2011), while other authors use Malaria parasites – more correctly, haemosporidian only morphology (Zehtindjiev et al. 2012), or a com- parasites – include six well-known genera of api- bination of both (e.g. Valkiūnas et al. 2009a,b; Falk complexans: Plasmodium (paraphyletic with res- et al. 2011). pect to mammal vs bird and lizard Plasmodium); The Biological Species concept is difficult to eval- Leucocytozoon, Haemoproteus and Parahaemoproteus uate in malaria parasites, and, instead, most research- (birds); Hepatocystis (mammals) and Polychromo- ers have used variations of the morphological and philus (bats), which, combined, infect all amniotes. phylogenetic species concepts. The morphological Less well-known genera that are not discussed in this species concept is essentially a similarity species con- review include Fallisia (Garniidae; Valkiūnas, 2005, cept (see Garnham, 1966; Valkiūnas, 2005); closely pp. 731) of birds and lizards and the plasmodiids related to this is the genetic similarity species concept unique to reptiles (Telford, 2009, pp. 160–199). (Martinsen et al. 2006). The phylogenetic species Although the anthropoid Plasmodium parasites concept has been applied by evaluating reciprocal have received the most attention, owing to their monophyly between cyt b sequences of named line- impact on human health, known malaria parasite ages (Martinsen et al. 2006). However, surprisingly biodiversity is broadest among bird species and popu- few studies of malaria parasites in wildlife have lations. This is primarily due to the long tradition explicitly addressed agreement across these species of taking blood smears and blood samples in field concepts: morphological similarity vs phylogenetic studies of birds, a practice that is less common among similarity (reciprocal monophyly) vs genetic simi- mammalogists. The earliest work on malaria con- larity. The most thorough is Martinsen et al. (2006), cerned avian parasites (reviewed in Valkiūnas, 2005, who evaluated five avian haemosporidian clades pp. 9–15), of which the c. 200 described species are using these three species concepts. In only one case based on morphological characters and/or host taxa. was there discordance across concepts, i.e. in which a In contrast, 1517 cytochrome b haplotypes have ‘species’ was paraphyletic; some species included been described worldwide, although only 122 are more than one cyt b sequence. associated with formally described species (MalAvi; Quality blood smears and sequence data have oc- Bensch et al. 2009). It is important to note here casionally provided morphological corroboration of that avian malaria researchers use different metrics ‘cryptic’ phylogenetic species (e.g. Valkiūnas et al. to define lineages, ranging from a single nucleotide 2010). One of the most compelling examples involved difference (Bensch et al. 2000, 2004; Hellgren, 2005) Leucocytozoon toddi (Sehgal et al. 2006), in which a to a combination of genetic differentiation and species described on the basis of a single morphotype host/geographic distribution (Ricklefs et al. 2005; was found to represent two genetic lineages present Svensson-Coelho et al. 2013). in different host genera, one in Buteo spp. and one in The diversity of avian malaria parasites probably Accipiter spp. This marked division caused a re- greatly exceeds the 1500 molecularly defined lineages evaluation of blood smears from these host groups, known at present. Based on current sampling, avian and morphological differences were in fact discovered malaria parasites are sister to mammalian Plasmodium between these two lineages (Valkiūnas et al. 2010). and are much more diverse than known mammal These studies were followed by others showing diver- parasites in terms of vector usage, levels of host sity within other Leucocytozoon morphospecies specificity, and geographic range. We have argued (Ishak et al. 2008; Krone et al. 2008). In an extensive that the most recent common ancestor of extant, treatment of Plasmodium relictum in birds, Beadell avian malaria parasite genera dates to only 9 MYA et al. (2006) found P. relictum to be composed of (Ricklefs and Outlaw, 2010) – certainly not much multiple, reciprocally monophyletic lineages that more than that (Bensch et al. 2013) – and that their are nonetheless quite closely related to one another. incredible diversity has been achieved primarily

through speciation associated with host-switching species descriptions from wild birds should be ‘dis- (Ricklefs et al. 2004, in review). couraged’ and their validity questioned, and that studies addressing ‘ecology and evolutionary biology based only on generic identification’ (p. 141) are HISTORICAL PHILOSOPHIES AND APPROACHES simply not adequate for use in parasitological re- The three most fundamental treatises on malaria search and examination. Thus, Valkiūnas emphasizes parasite diversity are Garnham’s, Valkiūnas’s the importance of training in classical parasitology for and Telford’s synthetic works (Garnham, 1966; species descriptions, and perhaps rightly so. Valkiūnas, 2005; Telford, 2009). In his discussion Telford’s monograph (Telford, 2009) focused of Classification and Evolution, Garnham (1966) entirely on reptilian parasites and, even then, mostly argued that not naming species, even putative ones, on species descriptions. However, because of the dis- leads to an underestimate of diversity and could parity between the number of known reptile haemo- actually thwart scientific understanding. He went on sporidians from morphology vs genetics, Telford to give some historical examples, and stressed that, emphasized that ‘until a broad spectrum of known given the paucity of characters available for de- species has been studied ...phylogenetic relation- scription and classification, any and all characters ships ...must remain based on morphological and should ‘be seized and used as liberally as possible’, life history traits’ (Telford, 2009, p. 1). Telford went particularly since it is not possible in the majority of on to say, as did Valkiūnas (2005), that although the cases to view all life-stages of a parasite (the gold genomic revolution will ultimately meet, one way or standard of species description in malaria parasites). another, with described morphospecies, molecular Indeed, the scarcity of characters had led most taxo- methods will not replace microscopy in the descrip- nomists (see below) to use characters associated with tion and naming of species. Indeed, Telford took the gametocytes (although other stages are quan- a somewhat defiant stance, understandable because tifiable in Plasmodium only), a point that Garnham traditional microscopy methods are often ignored: treated with some disdain, especially when coupled ‘morphology visible under light microscopy is with ‘host’ as a character. He went on to say that, essential in the definition and description of species, in gametocytes and even sporozooites (from the whatever relevance it may possess to genetic relation- dipteran host), morphological variation between ships among a fauna’ (italics added; Telford, 2009, parasite species is ‘trifling’. As pessimistic as these p. 12). Telford, like Valkiūnas (2005), advocated for statements may seem, Garnham presciently stated microscopy, and provided a detailed list of both that biochemical methods might ultimately replace direct (i.e. discrete measurements) and indirect (i.e. morphological description (see Garnham, 1966, host-cell deformation) characters that should be used pp. 64–72). to ‘describe’ species. Valkiūnas (2005) focused exclusively on malaria It is pertinent here to review the advantages of parasites in birds and took a more conservative ap- traditional microscopy methods before focusing on proach to the species question than did Garnham the use of molecular methods. Mixed infections are (1966). He also argued against the one-host, one- a formidable problem for molecular methods, be- parasite paradigm in favour of detailed descriptions cause these methods cannot readily distinguish two of parasite morphology in the peripheral blood of or more haplotypes of parasite; many researchers the vertebrate host with a deliberate de-emphasis of simply ignore such samples, but microscopy can be the host, except in some cases in which there is ex- used to distinguish parasites if they have been iden- perimental evidence of host preference (e.g. sub- tified previously. Molecular methods can also be inoculations; p. 232). However, in his taxonomic limited because of preferential amplification of some keys, host taxon is a primary ‘trait’ for species deter- parasite genetic lineages over others in the sample; mination in both the subgenus Parahaemoproteus some of this is due to higher levels of parasitaemia (p. 252) and the genus Leucocytozoon (p. 739). of one parasite vs another, but some is also due to Valkiūnas went on to say that morphometric charac- the molecular methods themselves. For example, ters are continuous and overlapping in, for example, ‘primers’ that are used to target specific parasite genes the ‘majority of [Leucocytozoon] species’, and indeed may have a greater affinity for some parasite lineages, that despite these overlapping characters, ‘combina- amplifying those over others, which can lead to under- tions of these in various species of haemoproteids are representation of rare lineages (Zehtindjiev et al. rather different and informative’ (pp. 76–78). These 2012); this problem can be addressed with ‘deep statements provide no explicit species criteria, but sequencing’ (Jarvi et al. 2013) which can uncover all Valkiūnas also suggested that it would be straight- sequence variants of a particular locus in any sample. forward to perform the necessary sub-inoculations Another problem is the mistaken assignment of gen- (albeit only in Plasmodium) to characterize morpho- etic lineages to morphospecies on databases (MalAvi logical characters of all parasite life-history stages and [Bensch et al. 2009] and GenBank; see below). not to rely solely on peripheral blood stages from However, one of the most challenging problems naturally infected birds. Indeed, he emphasized that involves the biology of infection; insect vectors may

inject into a vertebrate host sporozoites that may not An alternative to seeking congruence among multi- be able to reproduce inside that host, while molecular ple markers has been to use a cutoff level of sequence methods will nevertheless amplify DNA from that divergence to establish species boundaries. The cur- parasite. Ways around this problem include iden- rent value of this approach rests on the availability of tification (or not) of the parasite in a blood smear and cyt b sequence data and the desirability of leveraging sub-inoculations. Although the previous issues have these data for more than simply cataloguing novel been negative with respect to molecular methods, lineages. Based on empirical studies and compari- the majority of thorough examinations of concord- sons of well-characterized morphospecies and their ance between morphology and molecules have indeed counterpart cyt b sequences, researchers have pro- been positive: morphology matches molecules, at posed and subsequently ‘tested’ the idea that 5% least at the level of subgenera (e.g. Martinsen et al. sequence divergence in cyt b is a reasonable expec- 2006; Levin et al. 2012; Zehtindjiev et al. 2012). tation between morphologically differentiated species (Hellgren et al. 2007; reviewed in Valkiūnas et al. 2009a,b). The implication is that in the absence of morphological data, this cut-off could be used to RECENT DISCUSSIONS OF THE SPECIES QUESTION delimit species and estimate the diversity of haemos- AND DEVELOPMENT OF CRITERIA poridian parasites. However, advocates of this cut-off In 2011, two reviews addressed several major issues cautioned that ‘... a molecular criterion of over 5% concerning ‘species limits’ and ‘nomenclature’ in sequence divergence in cyt b gene for the identifica- malaria parasites: (i) the need for more well-trained tion of haemosporidian species should be developed parasitologists; (ii) reconciliation of morphological and applied carefully, preferably by linking molecular and molecular approaches; (iii) improving how we and microscopical data’ (italics added; Valkiūnas et al. specify lineages in molecular databases; and (iv) de- 2009a,b). In a recent treatment of cryptic and novel veloping criteria to define species (Perkins et al. lineages found in lizard parasites, the results were 2011; Littlewood, 2012). Resolving issues concern- consistent with the 5% ‘rule’ for cyt b divergence, in ing species delimitation is a necessary step towards that individual cyt b lineages differed by more than the goal of determining malaria parasite biodiversity 5%. The authors included morphological data (which and understanding haemosporidian evolution and were not informative), and two mitochondrial genes distribution. Past disagreement over these issues and one nuclear gene, across which there was concor- should not prevent us from coming to a consensus dance, making a strong case using reciprocal mono- that is transparent and open to modification. Both phyly for species delimitation (Falk et al. 2011). reviews mentioned above concluded with a call for more cooperation, particularly concerning the species THE NEED TO DELIMIT SPECIES, AND CRITERIA question, among research groups with different but FOR MOVING FORWARD overlapping expertise (i.e. morphometrics and genetics). Here, we suggest a framework within which we can Several criteria have been suggested to delimit define species or OTUs in a standardized way so that species in malaria parasites, including concordance the malaria parasite research community has a com- among multi-locus sequence data (Falk et al. 2011), mon language. The most straightforward and unam- absolute levels of sequence divergence (Bensch et al. biguous requirement under a phylogenetic species 2004), and fixed nucleotide differences (explored concept is phylogenetic concordance across multiple here) which may or may not involve sequence diver- loci (Perkins, 2000; Martinsen et al. 2006; Ramiro gence cut-off values (see below). Certainly, mono- et al. 2012). Concordance across multiple loci phyly across multiple gene trees would be the gold (including linkage disequilibrium between non- standard to delimit species, but because amplifying interbreeding populations; Bensch et al. 2004) is the non-mitochondrial parasite DNA has been difficult, acid-test for species limits, and the multiple, inde- this has not been possible in most analyses to this pendent genetic markers required for such analyses point. Moreover, the markers currently available should be accessible in the near future. However, from the nuclear and apicoplast genomes are not because such data are presently unavailable to most ideally suited to phylogenetic and population genetic researchers, and because most infections have been analyses because they are either too short and lack characterized to date by single mitochondrial gene informative, synapomorphic characters, or they pro- sequences, it is important to develop a method to cir- duce very long terminal branches (i.e. they contain cumscribe parasite lineages based on limited genetic more apomorphic than synapomorphic characters; information. We to address this issue here and offer a see Outlaw and Ricklefs, 2010), and in many cases, way to move forward. are not more informative than mitochondrial cyt b One goal of haemosporidian research is to de- (Perkins et al. 2007). Nevertheless, nuclear genes will velop distributional and evolutionary contexts for provide an increasing amount of data that will be individual lineages of parasites. Most malaria parasite useful for delimiting species. lineages from wildlife hosts are given arbitrary

Fig. 1. Bayesian maximum clade credibility tree of the avian malaria parasite cytochrome b gene tree based on 479 bp (BEAST 1.7.5; [Drummond and Rambaut, 2007]); posterior probabilities are highest at the terminals and all >0·80 are noted with circles of increasing size at the nodes). All genus-level clades are collapsed except for the focal Parahaemoproteus clade. All Parahaemoproteus clades that do not contain a sequence from a named lineage are also collapsed. The smallest monophyletic group to include all sequences (purple names) attributed to a named species is highlighted in blue and the speciﬁc epithet is adjacent to each clade. Embedded sequences attributed to other species are named in dark green. Two species whose sequences do not form clades are named in light green and in orange. Unnamed lineages are named in black. The full ﬁgure is available on request.

designations, like PTURDUS1 or Plasmodium sp. 1, parasites, will remain wholly inadequate. Garnham independently by each laboratory. In fact, based on (1966) understood the need to name species, even identity (100% GenBank match), 31 avian parasite when there were doubts, because ‘apart from the lineages have at least five names, and five of these have inconvenience [naming species in the absence of 10 or more names (July, 2013). Without standardized total evidence] causes ...it tends to inhibit acqui- names associated with taxonomic entities designated sition of knowledge by producing the impression by accepted criteria, our understanding of evolution that the life cycle of a parasite bearing the name and diversity, and, perhaps even more importantly, Plasmodium – such as the so-called Plasmodium of our ability to communicate effectively about these lizards – must be the same as the cycle of well-known

Table 1. Named Parahaemoproteus species in of July 2013) using the 479 base pairs of cyt b de- MalAvi, with the number of constituent lineages signated by Bensch et al. (2009)todefine lineages. based on Bayesian phylogenetic analysis of existing For the genus Parahaemoproteus (chosen because it cytochrome b data (Fig. 1) and the range of is the best sampled genus), we identified mono- pairwise sequence divergence values phyletic clades of all ‘named’ species for which there were at least two representative sequences in MalAvi Number of (purple taxon names, clades highlighted in blue, constituent in Fig. 1). Overall, there is a great degree of concor- lineages Number (including dance between named species and their constituent Parahaemoproteus of host unnamed Sequence clades, which is not surprising given that we used species species lineages) divergence named sequences to define clades and the fact that most named sequences were compared with those balmorali 3 12 0·001–0·039 belopolskyi 5 30 0·002–0·039 for which detailed morphological descriptions are cyanomitrae 1 9 0·002–0·035 available (e.g. Martinsen et al. 2006; Hellgren et al. haemobelopolskyi 1 4 0·002–0·006 2007). However, it is clear that issues remain with lanii 1 8 0·002–0·020 some named sequences. Named species that are em- – magnus 2 4 0·002 0·010 bedded (i.e. paraphyletic sequences) in one of these majoris 5 16 0·002–0·010 micronuclearis 3 10 0·002–0·024 clades are highlighted further in Fig. 1. Constituent minutus 2 9 0·000–0·010 sequences are not monophyletic for two named motacillae 1 6 0·002–0·010 species: two nucleofascialis in light green and six parabelopolskyi 2 28 0·002–0·041 tartakovskyi in orange. In four cases, named species – sanguinis 1 4 0·003 0·010 represented by a single sequence are embedded in named species clades: belopolskyi, balmorali, minutus and majoris. Because these are single representative members of the genus [i.e. human Plasmodium]’ sequences, we cannot assess whether their placement (Garnham, 1966, pp. 64). Thus, sequence data for is due to misidentification or whether they represent avian malaria parasites are abundant, and increasing ‘real’ species that have recently diverged. We cal- rapidly, and now researchers face the additional culated the range of pairwise distances in each of the problem of being provided with ambiguous criteria named species (Table 1). Variation in sequence diver- to determine novel lineages. In many cases, genetic gence within named species increases with sample divergence between malaria parasite lineages cannot size and exhibits a maximum value of 0·041, or about be linked to morphological variation (because blood 4%. The average intraspecific pairwise divergence smears are lacking; Bensch et al. 2004; Marzal et al. between cyt b sequences is 0·02 (range = 0·001–0·041; 2011; see also Pérez-Tris et al. 2005), or genetic diver- Table 1) whereas the average interspecific divergence gence does not match morphological variation, at value for all pairwise comparisons is 0·07 (range = least as indicated by species names (Beadell et al. 2006 0·013–0·092), which still includes some comparisons [P. relictum]). Hence, the utility of genetic lineages, that are clearly due to misnamed lineages (Table S1). but also of formally named species, is difficult to eval- The mean maximum values between the inter- uate in terms of representing independently evolving specific and intraspecific pairwise divergences differ lineages in the absence of a phylogenetic species significantly (P<0·001). However, it is clear from concept (Bensch et al. 2004). this analysis that misnamed or misclassified lineages One might ask whether it is reasonable to use hinder the broad applicability of a %-divergence cri- criteria for species delineation that have been de- terion; any conclusions drawn from this analysis veloped for other problematic organisms, such as presume that each name represents a biologically prokaryotes. Bacteriologists have developed a system, meaningful entity, which may not be the case. albeit not without controversy and uneven appli- In another case, a 3% or 4% rule would classify cation, that uses 16S rRNA sequence data to define three genetically divergent, partially allopatric line- lineages, with 3% divergence often being used to ages as a single species (Table 2, Fig. 2). Two of the distinguish species (Weisburg et al. 1991; reviewed lineages, DR03 and JA02, are found primarily in one in Forney et al. 2004). Of course, malaria parasites host species, the bananaquit (Coereba flaveola), but do not have a complete 16S rRNA sequence in their on different islands in the West Indies (Hispaniola genome, but researchers have argued for using cyt b and St. Vincent [DR03, having a disjunct distri- sequence divergence to delineate species (Hellgren bution] and Jamaica and the nearby Cayman Islands et al. 2007, but see Levin et al. 2012). Is it biologically [JA02]); LA07, although mostly associated with the meaningful to apply a %-divergence rule to cyt b to bananaquit, is widespread geographically, including delimit avian haemosporidian species? To address Mexico and Venezuela where it has been recovered the generality of a %-divergence rule, at least up to a from Icterus spp., but not including Jamaica, point, we reconstructed a Bayesian phylogeny of all Hispaniola, or St. Vincent; it is particularly common available avian malaria parasite lineages (n = 1300 as on Puerto Rico and Grenada. Fixed differences

Table 2. Genetic variation within lineages across 300 nt of mitochondrial cytochrome b

Number Number of Number of Haplotype Nucleotide Lineages of sequences segregating sitesa haplotypes diversityb diversityc

Total 67 5 4 0·607 0·006 JA02 7 0 1 0 0 DR03 28 1 2 0·071 0 LA07 32 0 1 0 0

JA, Jamaica; DR, Dominican Republic; LA, Lesser Antilles. a Number of polymorphic nucleotide sites in each lineage. b Uniqueness of haplotypes in each lineage. c Weighted sequence divergence between haplotypes.

Table 3. Fixation indices and demographic parameters. FST values were calculated in DNAsp (Librado and Rozas, 2009); coalescence and migrants per generation (NeM) were calculated in IMa (Hey and Nielsen, 2004; Hey, 2009) using the cytochrome b mutation rate from Ricklefs and Outlaw (2010)

a b c Lineage comparisons Fixed diﬀerences FST Coalescence time (years) NeM1 NeM2 JA02-DR03 3 0·988 471747 0·000 0·000 JA02-LA07 2 1·000 94255 0·001 0·125 DR03-LA07 3 0·988 166255 0·094 0·382

a Nucleotide diﬀerences that are unique to that lineage (based on 300–1000 nt). b Estimated number of migrants per generation from population 1 to population 2. c Estimated number of migrants per generation from population 2 to population 1.

phylogenetic species, provided we understand the geographic and host-taxonomic context of the constituent populations. Even if one argues that allo- patry, as in the case of DR03 and JA02 in Fig. 2, precludes testing reproductive compatibility to determine biological species, the populations are genetically distinct in cyt b (Tables 2 and 3), and the species tree generated from gene trees from markers (four) from all three genomes is highly supported (Fig. 2). None of these lineages is associated with a named species, and each is diﬀerent from the closest (BLAST) ‘named species’ by at least 5%.

Fig. 2. ‘Species’ tree of three lineages reconstructed using PROSPECTS gene trees from four loci using BEST (Liu, 2008). Characters used to describe species of malaria Support values are shown at nodes. Each of these lineages parasites, which are relatively few in number, are has >0·95 posterior probability in the cytochrome b gene likely to be conservative because these parasites are tree, with pairwise differences of 3 nt/1000 nt. highly specialized. This lack of morphological differentiation apparently has led to attaching the between these parasite populations in cyt b suggest same species name to more than one reproductively that they are isolated from one another despite their isolated entity (per Garnham, 1966). This, in turn, recent origin (Table 3). Blood smears from these has created cases of paraphyly and polyphyly of lineages, although available, have not yet been eval- named species, which we have identified in the avian uated, but the important point here is that even a few Parahaemoproteus phylogeny (Fig. 1). Conversely, nucleotide differences potentially could be associated named ‘species’ may harbour considerable species with species differences, and this is borne out by our diversity that cannot be identified because the analysis of these same populations using a multi- morphological characters are too few. Given the locus approach (Fig. 2; see also Braga et al. 2011). genetic diversity that we see among named haemo- Our results suggest that even these very young sporidian lineages, many named species of malaria populations of parasites can be designated as good parasite are like genera in birds, which are typically

Table 4. Suggested steps to develop diagnostic standards for species delimitation

Adopt a ‘tagging’ criterion for newly generated sequence data (e.g. 99% similarity or 1 fixed difference). Develop tagging system based on cyt b. Parse existing data into ‘tagged’ blocks of identical/similar sequences. Develop input/output pipeline in MalAvi. Develop standard procedure to send blood smear slides to experts. Facilitate consensus to identify experts who can describe new species and diagnose existing smears for species identification. Develop pipeline for processing blood smears. Ensure the field’s viability through training in classical parasitological techniques.

Fig. 3. Schematic of a proposed standardized procedure for identifying parasite lineages.

defined by morphological characters. Most avian for avoiding the naming of lineages independently of genera contain many species, but many of these were morphological characters. However, in many cases, misclassified to genus (i.e. misplaced in the taxo- molecular characters may be required to confirm the nomy) until molecular data began to sort this out, monophyly of named species and, in the absence of beginning primarily with Sibley and Alquist (1990). suitable morphological variation, sequence variation Just as ornithologists have done over the past 20 might be the only indication of species distinction. years, malaria parasitologists need to recognize that Of course, naming species is not the goal of most re- morphological descriptions are limited for haemo- searchers, and we outline an approach below that can sporidians, at least at the level of biological species, help to sort out the bewildering sequence diversity of and that other characters are required for species avian malaria parasites. delimitation and identification. At the same time, Developing a uniform standard for naming however, few young malaria parasitologists are being DNA sequences from haemosporidian parasites is trained in classical techniques including simple an important goal. The most general and useful blood smear preparation. Even with the abundance scheme at present is the MalAvi database (Bensch of genomic data on the horizon (S. Bensch, personal et al. 2009). Although MalAvi provides a system to communication), if the field lacks the expertise to tie name novel lineages, based on as little as a single genetic variation to life-history variation in the para- nucleotide difference, little headway has been made sites, opportunities to understand the broader con- since the creation of MalAvi in terms of applying its texts of parasite evolution and diversification will be information broadly to the species question. Most missed. malaria parasite researchers are less interested in naming new species than they are in having a protocol that allows them (1) to link their data to a broader CONCLUDING REMARKS database to determine the geographic distribution Our analyses show that the genetic variation within and host breadth of their lineages (but see Valkiūnas named haemosporidian species can be quite variable. et al. 2009a, b); and (2) to name an existing or novel This, of course, has been recognized by most malaria lineage in such a way that all researchers can easily taxonomists in recent years and it underlines the case identify that lineage. The first step would be to align

all cyt b sequence data currently on hand, a feature should be available to define species in a very robust of the MalAvi database (although data that do not way. However, in the meantime, given the rate at overlap MalAvi’ssequence‘standard’cannot beincor- which genetic lineages of malaria parasites are be- porated in this scheme), and organize MalAvi line- ing added to databases, we can usefully adopt a less ages into tagged blocks. Beyond that, one may wish to formal species definition. Moreover, because cyt b organize sequences into clades, potentially associated gene trees and parasite species trees bear a reasonable with named species, based on an arbitrary level of correspondence, cyt b is at present the most useful sequence similarity (e.g. 99%). When new data are tool that the field has to define species and/or OTUs. generated, a protocol could then include, for example: (1) BLAST to MalAvi and align to all tagged sequences; (2) if the sequence(s) match at a pre- SUPPLEMENTARY MATERIAL determined level (e.g. 99% sequence similarity, or no To view supplementary material for this article, fixed differences) with a previously tagged lineage, please visit http://dx.doi.org/S0031182014000560. clade of sequences, or named species, then that tag is applied to the new data and the researcher can easily determine the geographic and host range of the line- ACKNOWLEDGEMENTS age(s); (3) if the lineage is truly novel at some We wish to thank Dr Stephen Phillips for inviting us to arbitrary level of sequence divergence (e.g. >1%), write this review, and to acknowledge and thank members then it would be given a new tag. Figure 3 shows of our respective lab groups for critiques of previous drafts. how the process could work. However, recognizing Ravinder Sehgal and an anonymous reviewer provided ‘ ’ excellent criticism and comments that improved the that malaria species undoubtedly contain genetic manuscript. variation at most diagnostic loci, the application of any such criteria for distinguishing reproductively isolated entities should take into account the geo- REFERENCES

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