Org Divers Evol (2013) 13:111–126 DOI 10.1007/s13127-012-0115-1 ORIGINAL ARTICLE Contrasting phylogenetic patterns of anther smuts (Pucciniomycotina: Microbotryum) reflect phylogenetic patterns of their caryophyllaceous hosts Martin Kemler & María P. Martín & M. Teresa Telleria & Angela M. Schäfer & Andrey Yurkov & Dominik Begerow Received: 29 December 2011 /Accepted: 2 October 2012 /Published online: 6 November 2012 # Gesellschaft für Biologische Systematik 2012 Abstract Anther smuts in the genus Microbotryum often is a factor that should be taken into consideration in delimitat- show very high host specificity toward their caryophyllaceous ing species. Parasites on Dianthus showed mainly an arbitrary hosts, but some of the larger host groups such as Dianthus are distribution on Dianthus hosts, whereas parasites on other crucially undersampled for these parasites so that the question Caryophyllaceae formed well-supported monophyletic clades of host specificity cannot be answered conclusively. In this that corresponded to restricted host groups. The same pattern study we sequenced the internal transcribed spacer (ITS) was observed in the Caryophyllaceae studied: morphological- region of members of the Microbotryum dianthorum species ly described Dianthus species did not correspond well with complex as well as their Dianthus hosts. We compared phy- monophyletic clades based on molecular data, whereas other logenetic trees of these parasites including sequences of anther Caryophyllaceae mainly did. We suggest that these different smuts from other Caryophyllaceae, mainly Silene,withphy- patterns primarily result from different breeding systems and logenies of Caryophyllaceae that are known to harbor anther speciation times between different host groups as well as smuts. Additionally we tested whether observed patterns in difficulties in species delimitations in the genus Dianthus. parasites are due to shared ancestry or if geographic separation Keywords Species diversification . Host specificity . Host range . Microbotryum . Caryophyllaceae . ITS Electronic supplementary material The online version of this article (doi:10.1007/s13127-012-0115-1) contains supplementary material, which is available to authorized users. Introduction M. Kemler : A. M. Schäfer : A. Yurkov : D. Begerow Lehrstuhl für Evolution und Biodiversität der Pflanzen, AG Geobotanik, Ruhr-Universität Bochum, Parasitic organisms (i.e., symbionts that withdraw nutrients; Bochum, Germany sensu Hall 1974) occur in many evolutionary lineages (Hawksworth 2001; Poulin and Morand 2000) and account M. P. Martín : M. T. Telleria Departamento de Micología, Real Jardín Botánico-CSIC, for much of biological diversity (Price 1980). Species rich- Plaza de Murillo 2, ness and radiation have been mainly attributed to host shifts 28014 Madrid, Spain leading to reproductive isolation and adaption to new hosts (Zietara and Lumme 2002; Giraud et al. 2010). Even though Present Address: M. Kemler (*) all parasites are restricted to a certain range of hosts they Centre of Excellence in Tree Health Biotechnology; infect, there are huge differences in the amount of hosts used Forestry and Agricultural Biotechnology Institute (FABI), between parasites, even in closely related organisms (De University of Pretoria, Wet et al. 2008). The range of potential hosts a parasite Pretoria 0002, South Africa e-mail: [email protected] can use has an impact on many aspects of the parasites ecology and evolution, e.g., distribution range and fitness Present Address: in a given locality. Broad host spectra enable parasite spe- A. Yurkov cies to spread in a greater geographic range because of Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures, differences in host distributions, thereby additionally en- Braunschweig, Germany hancing the chance for host switches (Janz and Nylin 112 M. Kemler et al. 2008). Parasites with narrow host ranges have a higher 2005, 2008), and recently new species have been proposed fitness on their host than multitrophic parasites occurring (Denchev et al. 2009), but these studies also indicate that host on the same host as they are assumed to be better adapted to specificity of the Dianthus parasites may be rather low com- their hosts (Lajeunesse and Forbes 2002). pared to other anther smuts. Thereby, the number of parasite Interestingly, phylogenetic methods have shown that host specimens on different Dianthus species considered in previ- specificity seems much higher in many parasitic groups than ous studies has not been very extensive, making it difficult to has previously been assumed based on morphological data, assess whether this pattern of low host specificity is common but infection studies show that many parasites possess the in anther parasites of Dianthus or these observations are ability to infect a multitude of hosts under artificial conditions sampling artifacts. As low taxon sampling may have serious (Poulin and Keeney 2008). This suggests that host specificity effects on phylogenetic errors (Hillis 1996, 1998), the inten- is a much more complex phenomenon than was previously tion of this study was to infer relationships between parasites assumed and involves more factors than just the co-evolution on different Dianthus species based on a larger taxon sam- between host and parasite. One aspect of host parasite inter- pling and to compare the phylogenetic pattern obtained with actions that mediates this complexity is the spatial context, the pattern from other Caryophyllaceae, mainly from Silene, including geographic barriers limiting distribution of hosts parasites. Variability of the internal transcribed spacer nrDNA and parasites. As indicated above, host specificity can influ- (nrITS including internal transcribed spacers 1 and 2 as well as ence the geographic ranges of parasite species, but geographic the 5.8S gene) sequences was analyzed in both parasites and ranges are also shaped by life cycles and dispersal abilities of hosts using phylogenetic approaches; additionally host spec- both hosts and parasites (Poulin et al. 2011). Due to differen- ificity and geographical distribution were examined. This was tial selection of distinct populations in different areas done to answer three questions: (1) Is low host specificity in (Thompson 2005), this complexity of factors over different Dianthus parasites common or are previous observations arti- spatial scales will ultimately also influence the genetic struc- facts of a sampling bias? (2) Is there a correlation between host ture of the species involved. Hence, a parasites’ host range and specificity and geographic distribution in Dianthus and Silene host specificity are shaped by the combination of continuously parasites? (3) Is there a correlation between genetic variation interacting spatial and evolutionary factors. of parasites of Dianthus and other Caryophyllaceae, mainly Members of the smut fungal genus Microbotryum Lév. Silene, with host genetic variation? are parasites on a large range of eu-dicotyledonous host plants (Vánky 1998). The different species form their spores mainly in the flowers of their hosts, but there are also Materials and methods species that sporulate in stems [e.g., Microbotryum parla- torei (A.A. Fisch. Waldh.) Vánky] or leaves [e.g., Taxonomy of Microbotryum and Dianthus Microbotryum marginale (DC.) Vánky]. Besides a great host spectrum within the genus, the most well-known The DNA sequences from the fungal specimens of this study Microbotryum species are the anther smuts of the are listed in Table 1. The taxonomy used in the study follows Caryophyllaceae that infect different species of the genera Denchev (2007), Denchev et al. (2009), Vánky (1994), Lutz et Dianthus L., Silene L., Saponaria L., Stellaria L. and other al. (2005, 2008)andPiątek et al. (2012). Specimens from members of that plant family. Many caryophyllaceous host Dianthus were named Microbotryum dianthorum s.l. as there genera, except for Silene, seem to have been colonized only are no ITS sequences available for the new species recently once with subsequent radiations within these groups (Le described by Denchev et al. (2009) and the different host Gac et al. 2007;Lutzetal.2005, 2008). Traditionally, species can be colonized by more than one parasite species. species delimitations in the caryophyllaceous anther smuts The DNA sequences from the host plants of this study are have relied on the host plant (Fig. 1a), spore morphology listed in Table 2. The taxonomy used in the study follows (Fig. 1b) and spore color, but have been debated for a long Frajman et al. (2009a), Frajman and Oxelman (2007), time (Liro 1924; Lindeberg 1959). Recent molecular studies Oxelman et al. (2001) and Tutin and Walters (1993). have demonstrated very high host specificities in the para- sites of studied Silene species (Le Gac et al. 2007; Lutz et al. Molecular analyses 2005, 2008), and within this group a one-to-one relationship between parasites and hosts is a common finding, e.g., In order to extract genomic DNA, the DNeasy™ Plant Mini Microbotryum chloranthae-verrucosum M. Lutz, Göker, Kit (QIAGEN, Germany/ USA) or the EZNA™ Fungal Piątek, Kemler, Begerow & Oberw. on Silene chlorantha DNA Miniprep Kit (without adding 2-mercaptoethanol in Hort ex Fenzl (Lutz et al. 2005). tissue lysing; Omega Biotek, USA) was used. The ITS Anther smuts on species of Dianthus seem to be separated region of the parasites was amplified by polymerase chain into several groups as well (Le Gac et al. 2007;Lutzetal. reaction