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spore size is the morphological characteristic that most closely correlates with rDNA clades of phyloge- netic trees. This study demonstrates that traditional.
Mycologia, 103(1), 2011, pp. 36–44. DOI: 10.3852/09-314 # 2011 by The Mycological Society of America, Lawrence, KS 66044-8897

Phylogeny of Pilobolaceae K. Michael Foos1 Nicole L. May Dale L. Beach2 Markus Pomper

7–9 species (Grove 1934, Hu et al. 1989, Zycha et al. 1969). These fungi, primarily coprophilous saprobes, are ubiquitous with the exception of Utharomyces, which is restricted to tropical habitats. Zygospores have been reported in Pilaira and Pilobolus but not in Utharomyces. Because zygospores are rare in species of this family the taxa usually are characterized by asexual reproductive structures. All three genera have sporangia containing large numbers of sporangiospores, a persistent dark pigmented sporangial wall and papillate to broadly rounded columella. Sporangiophores are large and phototrophic; Pilobolus and Utharomyces possess subsporangial swellings and trophocysts (Kirk and Benny 1980). Mechanisms of sporangiospore discharge also distinguish the genera. Sporangiophores of Pilaira lack subsporangial swellings and are the least complex of the group. Sporangiophores of Pilaira and Utharomyces are relatively short during early growth, but they elongate rapidly as they mature, aiding dissemination of sporangiospores. Pilobolus disseminates sporangiospores by ballistic discharge caused by the elevated pressure within the subsporangial swelling of the sporangiophore. Pilaira and Utharomyces can be maintained for years on common laboratory media, but with the exception of a few isolates (Kubo and Mihara 1986) Pilobolus is particularly difficult to culture on artificial media or even on dung agar. In most instances chelated iron is an essential ingredient, but even media enriched with chelated iron fail to support the growth of many isolates. Some isolates do not survive the transfer from dung. Of the strains that can be cultivated on artificial media most survive through only a few sequential transfers. Furthermore only rare isolates survive long term preservation, resulting in few isolates of Pilobolus in culture collections worldwide, most of which are duplicates held in multiple collections. Therefore it is imperative to develop a rapid, reproducible and reliable method of identification and phylogenetic determination for these organisms that are difficult or impossible to cultivate. White et al. (2006) noted a need for additional data from understudied and unculturable taxa to understand the evolutionary history of the Zygomycota. The genera of Pilobolaceae are such taxa, often studied because of their spore discharge mechanisms, but whose evolutionary history has not been examined closely. Historically Pilobolaceae has been categorized as one of 12 families of Mucorales. However recent studies indicate that this classification is highly

School of Natural Science and Mathematics, Indiana University East, Richmond, Indiana 47374

Kathy B. Sheehan Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347

Donald G. Ruch Department of Biology, Ball State University, Muncie, Indiana 47306

Abstract: The three genera traditionally classified as Pilobolaceae have been identified on the basis of morphological characteristics. In the absence of distinctive morphological differences phylogenetic techniques have proven to be superior for developing phylogenies. Molecular techniques have been used primarily for studies of higher fungi; there are few investigations of the Zygomycota using genetic sequences for classification. DNA sequences coding for three regions of rRNA were used to investigate phylogenetic relationships of the three genera traditionally considered within the Pilobolaceae. Evidence indicates that Pilaira should be removed from Pilobolaceae and the family redescribed. Sporangiospore size is the morphological characteristic that most closely correlates with rDNA clades of phylogenetic trees. This study demonstrates that traditional morphological characteristics alone are not adequate to differentiate species of Pilobolus. Key words: DNA, ITS, LSU, mitochondria, Pilaira, Pilobolaceae, Pilobolus, ribosome, rRNA, SSU, Utharomyces, 5.8S, 18S, 23S INTRODUCTION

Family Pilobolaceae (Eukaryota, Fung, Fungi incertae sedis, Basal fungal lineages, Mucoromycotina, Mucorales) traditionally contains three genera, Pilobolus Tode ex Fr., Pilaira van Tiegh. and Utharomyces Boedijn (Kirk et al. 2001). Pilaira is reported to have five species, Utharomyces a single species and Pilobolus Submitted 20 Dec 2009; accepted for publication 22 Jul 2010. 1 Corresponding author. E-mail: [email protected] 2 Current address: Department of Biology and Environmental Sciences, Longwood University, Farmville, VA 23909.

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FOOS ET AL.: PILOBOLACEAE artificial (O’Donnell et al. 2001, Benny 2005) and should be re-evaluated as additional molecular studies are reported. Correct identification to species is a prerequisite for creation of a correct phylogeny. In the case of Pilobolus identification to species is particularly problematic. Species descriptions traditionally have been based on morphological characteristics, such as length of the sporangiophore and shape, size and pigmentation of the sporangiospores. However several factors make many morphological characteristics used for species identification unreliable. Even though the size and shape of the sporangiospores have been found to be stable within species (Foos and Jeffries 1988), species descriptions typically give such a large range of sporangiospore sizes that this character provides insufficient information to make an identification to species. For example Grove (1934) describes the spore lengths of P. longipes, P. kleinii and P. sphaerosporus as 12–15 mm, 11–20 mm and 10–20 mm respectively. In such cases where sporangiospore dimensions overlap spore shape (globose or elliptical) becomes the principal characteristic for differentiating species. Wide variations of the dimensions of other structures (i.e. sporangial diameters and sporangiophore lengths) used in species descriptions emphasize the need for the identification of other useful taxonomic characters. Taylor et al. (2006) observed that phylogenetic species recognition lets biologists sort individual organisms into species and then find which of many variable phenotypic characters of morphological species correlate with phylogenetic species. Second, a typical sample of fresh dung contains multiple species of Pilobolus (Foos 1997). Similar structures from multiple species growing intermingled on the surface of dung are nearly indistinguishable. Attempting to identify a species found on a dung sample easily could lead to misidentification, necessitating the generation of pure cultures for accurate identification. The challenges in transferring and culturing Pilobolus in laboratory media are formidable and have been discussed earlier. Finally, isolates that do survive multiple transfers onto culture media often exhibit structures that differ in size from those of the original isolate growing on the original dung sample. For example we compared the lengths of sporangiophores of P. longipes growing on an original dung sample and the growth derived from single sporangium transferred to dung agar. The culture on dung agar produced sporangiophores an order of magnitude shorter than the sporangiophores growing on the original dung sample. Reliance on such malleable morphological characteristics is likely to lead to incorrect identification of isolates and has lead to the misidentification of

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existing cultures, such as the American Type Culture Collection isolate P. kleinii (ATCC 14499), which is cross listed by the Centraalbureau voor Schimmelcultures as P. crystallinus (CBS 272.31). In the absence of stable morphological characteristics for identification a more reproducible basis for identification is required. The current study was designed to employ genetic data to clarify relationships among the three genera traditionally included in Pilobolaceae and to develop a reliable means to distinguish among species of Pilobolus. O’Donnell et al. (2001) completed a comprehensive study of the Mucorales with partial nucleotide sequences of the nuclear 18S ribosomal RNA (SSU), nuclear large subunit 28S ribosomal RNA (LSU) and EF-1a gene exons. The phylogeny of Mucorales also was studied by White et al. (2006), who used the combined rRNA operon (18S + 28S + 5.8S genes) to infer relationships. Millar et al. (2007) tested the 18S and ITS fragments and found them to be highly reliable in providing the correct identification of filamentous fungi and yeasts. Seif et al. (2005) suggested that mitochondrial DNA sequences could be used to examine comparative relationships among fungi. Other genes have been used to study the genetic diversity of fungi, but few studies have examined the individual families within Mucorales. Based on the successful application of mitochondrial and nuclear genes expressing ribosomal RNAs, we anticipated that genetic factors could be used to identify isolates of members of Pilobolaceae. The conservation of the rRNA coding regions implied that the same primers used to produce amplicons in other fungi (White et al. 1990) could be used throughout the family, as demonstrated by O’Donnell et al. (2001) for Mucorales. In this study we determined that nuclear encoded ribosomal DNA (rDNA) is superior to mitochondria-encoded rDNA for investigating the phylogenic relationships within Pilobolaceae. MATERIALS AND METHODS

Isolates of Pilobolus, Pilaira and Utharomyces were obtained from the American Type Culture Collection (ATCC), National Center for Agricultural Utilization Research (NRRL) and University of Alberta Microfungus Collection and Herbarium (UAMH). Other isolates were collected with techniques described by Foos et al. (2001). All three isolates of Pilobolus longipies were collected in Indiana, and the isolate of Pilobolus roridus was collected in Wyoming. The multiple isolates provide replicates that could reveal any variation among the strains of a species (TABLE I). All cultures were maintained at 22 6 2 C, with alternating 12 h light and dark periods of 2000 lux, cool white fluorescent illumination. Isolates were cultured on a synthetic hemin medium (Levetin and Caroselli 1976) in

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TABLE I. Voucher number, species, source and GenBank accession numbers for DNA sequences used in this study (SSU— fragment amplified by NS1–NS8 primers, ITS—fragment amplified by ITS5 and ITS4 primers, LSU—mitochondrial fragment amplified by ML7 and ML8 primers). All specimens represent independent isolates of each species Voucher number

Species

Source

SSU GenBank

ITS GenBank

IUE340 IUE409 IUE563 IUE410 IUE519 IUE520 IUE518 IUE521 IUE566 IUE701 IUE415 IUE567 IUE702 IUE703 IUE573 IUE574 IUE564 IUE605 IUE606 IUE607

Pilobolus longipes Pilobolus longipes Pilobolus longipes Pilobolus umbonatus Pilobolus umbonatus Pilobolus umbonatus Pilobolus sphaerosporus Pilobolus sphaerosporus Pilobolus kleinii Pilobolus kleinii Pilobolus roridus Pilobolus crystallinus Pilobolus crystallinus Pilobolus crystallinus Pilaira anomala Pilaira anomala Utharomyces epallocaulus Utharomyces epallocaulus Utharomyces epallocaulus Utharomyces epallocaulus

IUE 340 IUE 409 IUE 563 NRRL 6349 UAMH 7297 UAMH 7298 UAMH 3070 UAMH 1312 ATCC 14499 ATCC 36185 IUE 415 ATCC 11505 ATCC 46942 ATCC 36186 ATCC 36774 ATCC 36779 ATCC 42705 NRRL 3168 NRRL A-15807 NRRL A-21843

DQ211053 DQ211054 EU595654 EU595657 DQ211050 DQ211051 DQ211052 EU595653 EU595655 EU595656 EU595649 EU595650 EU595651 EU595652 EU595658 EU595659 EU595660 EU595661 EU595662 EU595663

FJ160950 FJ160951 FJ160952 FJ160955 FJ160956 DQ058412 DQ059382 FJ160953 FJ160954 FJ160957 FJ160948 FJ160947 FJ160958 FJ160949 FJ160942 FJ160941 FJ160943 FJ160944 FJ160945 FJ160946

disposable plastic Petri dishes sealed with parafilm. Mature sporangia were collected directly from sporangiophores with microforceps or from the lids of the Petri dishes with inoculating needles. Sporangia were placed in 0.2 mL microcentrifuge tubes containing 20 mL sterile collecting water (containing 3% penicillin, 3% streptomycin and 1% Tween 20), labeled and stored at 4 C. DNA was extracted from sporangiospores with the MoBio UltraClean Soil DNA Isolation Kit (Mo-Bio Laboratories Inc., Carlsbad, California) with the following modifications. The collecting water and sporangia from a single microcentrifuge tube were added to a 1.7 mL tube provided in the kit. Spores were heat treated at 70 C for 10 min before breaking sporangiospore walls with a FastPrep FP 120 instrument (Thermo Scientific, Waltham, Massachusetts), set at 6.5 for 45 s for one cycle. After extraction the total genomic DNA was stored in water at 220 C. Total DNA was amplified with AmpliTaqH Gold DNA polymerase (Applied Biosystems, Foster City, California) and a dNTP mix (Promega Corp., Fitchburg, Wisconsin). Thermal cycling was conducted in a Perkin Elmer GeneAmpH PCR System 2400. PCR reaction conditions for thermal cycling were 94 C for 5 min, followed by 36 cycles of 94 C for 1 min, 50 C for 1 min 30 s, 72 C for 2 min, followed by an extension at 72 C for 7 min. PCR products were purified with the QIAquickH PCR Purification Kit (QIAGEN Inc., Valencia, California). Primers selected to amplify and sequence specific fragments of DNA were described originally by White et al. (1990). Primers ML7 and ML8 were used to amplify a fragment of mitochondrial DNA coding for a portion of the 23S fragment within the large subunit (LSU) of rRNA to determine the extent that mitochondrial ribosomal RNA

LSU GenBank EF565865 EF565866 EF565873 EF565867 EF565870 EF565871 EF565869 EF565872 EF565875 EU595646 EF565868 EF565876 EU595647 EU595648 EF565877 EF565878 EF565874 EF565879 EF565880 EF565881

sequences differentiate members of Pilobolaceae. Primers NS1–NS8 were used to amplify the nuclear 18S fragment coding for the small subunit (SSU) of rRNA to compare with other phylogenetic studies of the family. Primers ITS5 and ITS4 were used to amplify the nuclear DNA fragment coding for the entire ITS fragment, including the 39 end of the SSU, the 5.8S, both of the internal transcribed spacers and the 59 end of the LSU. PCR-amplified DNA fragments were electrophoresed on 1% agarose gels in 13 TBE buffer (50 mM Tris-HCl, 50 mM boric acid and 1 mM EDTA) containing ethidium bromide and viewed with a ChemiImagerTM 4400 Imaging System (Alpha Innotech, San Leandro, California). A 100 bp DNA ladder (Takara Mirus Bio, Madison, Wisconsin) was used as a size marker. PCR products were sequenced with a BigDyeH Terminator 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California). Each sequence reaction contained 2 mL H2O, 3 mL 53 buffer, 1 mL BigDye, 2 mL 10 mM primer and 2 mL DNA. Thermal cycling conditions for sequencing were 25 cycles of 96 C for 10 s, 50 C for 5 s and 60 C for 4 min. An Applied Biosystems 3700 automated fluorescence system at the Indiana Molecular Biology Institute was used to sequence amplicons. Sequences were examined and compared with CodonCode Aligner (CodonCode Corp., Dedham, Massachusetts) containing PHRED and PHRAP (Ewing and Green 1998, Ewing et al. 1998) for base calling, sequence comparisons and sequence assembly. Contigs created in CodonCode were oriented with BLAST (Altschul et al. 1990) and aligned with Clustal W (Thompson et al. 1994, 1997). Where sequencing reactions produced ambiguous or mixed results, PCR products were cloned and the resulting plasmid inserts were sequenced. A TA-Cloning Kit (Invitro-

FOOS ET AL.: PILOBOLACEAE

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TABLE II. Lengths (bp) of the various rDNA fragments analyzed in this study (SSU—fragment amplified by NS1–NS8 primers, ITS—fragment amplified by ITS5 and ITS4 primers, LSU—mitochondrial fragment amplified by ML7 and ML8 primers). Voucher numbers refer to specimens in TABLE I Sections of ITS sequences Voucher number

Species

SSU

LSU

ITS

18S

ITS1

5.8Sa

ITS2

28S

IUE340 IUE409 IUE563 IUE410 IUE519 IUE520 IUE518 IUE521 IUE566 IUE701 IUE415 IUE567 IUE702 IUE703 IUE573 IUE574 IUE564 IUE605 IUE606 IUE607

Pilobolus longipes Pilobolus longipes Pilobolus longipes Pilobolus umbonatus Pilobolus umbonatus Pilobolus umbonatus Pilobolus sphaerosporus Pilobolus sphaerosporus Pilobolus kleinii Pilobolus kleinii Pilobolus roridus Pilobolus crystallinus Pilobolus crystallinus Pilobolus crystallinus Pilaira anomala Pilaira anomala Utharomyces epallocaulus Utharomyces epallocaulus Utharomyces epallocaulus Utharomyces epallocaulus

1763 1763 1763 1764 1764 1764 1764 1764 1764 1763 1762 1761 1763 1763 1761 1761 1748 1748 1748 1748

370 370 370 370 370 370 370 370 370 370 370 370 370 370 371 371 370 370 370 370

688 688 688 706 707 707 694 694 694 704 694 695 689 657 667 667 624 624 624 624

39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39

226 226 226 180 180 180 234 234 234 236 213 215 207 213 214 214 189 189 190 190

153 153 153 152 152 152 152 152 152 152 152 152 152 152 155 155 154 154 154 154

231 231 231 296 297 297 230 230 230 238 251 250 252 214 220 220 203 203 202 202

39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39 39

a

Terminal bases for 5.8S fragments were AAAACAACTT and TGTTTCAGT.

gen, Carlsbad, California) was used to clone PCR fragments into the precut cloning site of pCR2.1. Ligations were completed with the manufacturer’s protocol and transformed into chemically competent TOP10 cells (Invitrogen, Carlsbad, California). Bacterial colonies with plasmid pCR2.1 containing an insert were identified as white colonies in a blue-white screen. DNA was prepared from these colonies and submitted for sequencing with the same primer sets as described for direct sequencing of amplicons. Phylogenetic analyses were conducted and trees constructed with MEGA 4 (Tamura et al. 2007). DNA sequences used as outgroups were obtained from GenBank. The evolutionary history was inferred with neighbor joining (Saitou and Nei 1987). The bootstrap consensus trees inferred from 2000 replicates was taken to represent the evolutionary history of the taxa analyzed (Felsenstein 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (2000 replicates) is shown next to the branches. The trees are drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed with maximum composite likelihood (Tamura et al. 2004) and are in the units of the number of base substitutions per site. All positions containing alignment gaps and missing data were eliminated in pairwise sequence comparisons (pairwise deletion option).

Sporangiospores suspended in water were examined, measured and photographed with an Olympus BH-2 light microscope. Kodachrome 64, 35 mm slides were digitized with a Nikon Coolscan V. Digitized images were processed for publication with ArcSoft Photostudio5, converted to eight-bit grayscale, adjusted in contrast and brightness, cropped and superimposed with a black bar representing 10 mm. All images are reproduced at the same scale. RESULTS

DNA encoding a portion of mitochondrial LSU rRNA.— Amplicons were obtained with ML7 and ML8 primers. The resulting nucleotide sequences used for analysis correlated with published fungal mitochondrial rDNA sequences. Ribosomal DNA fragments produced from Pilobolus, Pilaira and Utharomyces differed by eight polymorphisms and one indel. Nucleotide sequences of the mitochondrial LSU fragments from isolates of the same genus were identical for Pilaira and Utharomyces, but LSU sequences from P. umbonatus differed from other Pilobolus isolates by a single nucleotide change. The LSU sequences of the three isolates of P. umbonatus were identical (TABLE II). Sequence alignments were used to construct a phylogram inferring the relationships of the genera.

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FIG. 1. Phylogram inferred from sequences of DNA coding for portions of the LSU (23S) mitochondrial rRNA of Pilobolaceae with neighbor joining. A total of 371 positions were in the final dataset. Bar 5 0.002 base substitutions per site. Voucher numbers correlating with TABLE I are included for each isolate.

The phylogram (FIG. 1) shows the separation of Utharomyces, Pilaira and Pilobolus. The orthologous sequence from Rhizopus oryzae (AY863212) from GenBank was used as outgroup. Analyses of the mitochondrial LSU support the current circumscription of described genera. Mitochondria-encoded LSU is sufficient to distinguish between genera but not species. Nuclear DNA encoding the SSU rRNA.—Sequences of the SSU with primers NS1–NS8 produced overlapping segments of DNA sufficient to generate a single contig for each species. The lengths of the fragments of DNA coding for nuclear SSU genes for Utharomyces, Pilaira and Pilobolus were characteristic for each genus (TABLE II). Small variations were observed within Pilaira and Utharomyces; Pilobolus demonstrated greater heterogeneity. The phylogram (FIG. 2) inferred from the nuclear DNA coding SSU fragments show distinct separation of the three genera with Pilobolus and Utharomyces in one clade and Pilaira as a separate clade. Bootstrap values suggested the orthologous SSU region of Rhizopus oryzae (GU126375) as outgroup for the phylogram. The phylogram (FIG. 2) shows isolates of Pilobolus separated into two main clades corresponding with differences in spore sizes of the species in each group. Large-spore (. 10 mm long) producing species (FIG. 3A–D; P. longipes, P. kleinii and P. sphaeros-

FIG. 2. Phylogram inferred from sequences of DNA for SSU (18S) rRNA of Pilobolaceae with neighbor joining. A total of 1828 positions were in the final dataset. Bar 5 0.005 base substitutions per site. Voucher numbers correlating with TABLE I are included for each isolate.

porus) comprise a major clade and those of smallspore (, 10 mm long) producing species (FIG. 3E–H; P. crystallinus, P. roridus and P. umbonatus) comprise a second major clade. The designations of large and small spores, consistent with the isolates under study, were derived from spore descriptions by Grove (1934), Zycha et al. (1969) and Hu et al. (1989). Isolates of each species previously identified on the basis of morphological characteristics clustered together in multiple clades strongly supported by bootstrap values. The molecular phylogeny was similar to the classification of morphological species. Integration of these data with previous studies.—Because the data from this study represent a detailed analysis of Pilobolaceae the findings were integrated into a larger dataset containing a broad selection of mucoralean families. The 18S rDNA sequences determined in this study were combined with a similar set of sequences used by O’Donnell et al. (2001) to establish phylogenetic relationships within Mucorales. A phylogram (not shown) constructed from combined data of O’Donnell et al. (2001), and this study was consistent with the previously published study and resulted in the separation of Pilaria from Pilobolus and Utharomyces. Nuclear DNA encoding the ITS region of rRNA.— Amplicons produced with ITS5 and ITS4 primers comprise the ITS region containing the entire ITS1, 5.8S and ITS2 regions of the rDNA transcript as well as flanking SSU and LSU regions. We provided the composition of the ITS regions among the three genera of Pilobolaceae (TABLE II). Isolates of Pilaira had identical sequences. Utharomyces generated prod-

FOOS ET AL.: PILOBOLACEAE

FIG. 3. Sporangiospores of select isolates of Pilobolus species. A. Pilobolus longipes IUE409. B. P. kleinii IUE701. C. P. sphaerosporus IUE521. D. P. kleinii IUE566. E. P. crystallinus IUE703. F. P. crystallinus IUE702. G. P. roridus IUE415. (H) P. umbonatus IUE410. Bars 5 10 mm.

ucts with the same overall length of the ITS region, however variation in the length of the spacer regions ITS1 and ITS2 were observed. The lengths of amplicons from isolates of Pilobolus were not uniform (TABLE II). The phylogram (FIG. 4) generated from the ITS region indicates phylogenetic relationships among Pilobolaceae, clearly differentiating the genera. As observed for the SSU sequence, Pilaira was distantly separated from Utharomyces and Pilobolus. Utharomyces and Pilobolus occurred in separate clades (FIG. 4). A sequence from Pilobolus crystallinus (NBRC 8561), the only ITS sequence from Pilobolus available from any lab other than ours, was included in the analysis of the ITS region (NBRC 2005). The ITS sequence of NBRC 8561 differed by several nucleotides from IUE 703 yet formed a clade with isolate IUE 703 separate from the other P. crystallinus isolates. Bootstrap values suggested the orthologous ITS region of Rhizopus oryzae (AM933544) as outgroup for the phylogram. DISCUSSION

Molecular phylogenetics has made significant changes to the taxonomy of microbial organisms. Identification and description of organisms on the basis of molecular data provide a statistically reliable and readily accomplished methodology superior to cladistic analysis based on a few morphological or biochemical characteristics for determining species. In the fungal kingdom comparative analysis of molecular data with phylogenies based on morphological characteristics suggests that several organisms

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FIG. 4. Phylogram inferred from sequences of DNA coding for ITS rRNA of Pilobolaceae with neighbor joining. A total of 806 positions were in the final dataset. Bar 5 0.05 base substitutions per site. Voucher numbers correlating with TABLE I are included for each isolate.

must be redescribed in light of the increased resolution of DNA sequences (Stajich et al. 2009, O’Donnell et al. 2001). The molecular phylogenetic approach has been sparingly applied to the Zygomycota. This report presents a detailed phylogenetic analysis of Pilobolaceae with elements of the nuclear encoded rRNA genes and the mitochondrial LSU gene as described. Phylograms constructed from the three genetic characters (mitochondrial LSU rRNA, nuclear ITS region and nuclear SSU rRNA) separated the genera, placing each genus in a separate clade with strong bootstrap support (FIGS. 1, 2, 4). The phylogram inferred from the nuclear ITS region (FIG. 4) is similar to the one inferred from the SSU region (FIG. 2) and clearly segregates the three genera and within Pilobolus separates the species with strong bootstrap support. The inclusion of multiple genetic loci is important for the resolution of species within the population. Often different single traits will lead to significantly different trees. When SSU and ITS sequences are compared the topologies of the resulting phylograms are not always conserved (Abe et al. 2006). In light of the similarity of the SSU and ITS, especially considering the variation observed within the ITS regions, the phylograms produced by these analyses supported the species designations used in this paper. The phylogram inferred from the mitochondrial LSU delimited by the ML7 and ML8 primers (FIG. 1)

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has a much lower resolution than phylograms inferred from nuclear ribosomal DNA sequences. The isolates used in this study segregated into three clades corresponding to the three genera. The sequences obtained for the mitochondrial LSU unexpectedly contained little phylogenetic information. The findings of the present study support observations by Seif et al. (2005) who suggested that mitochondrial DNA sequences could be used to examine relationships at the upper phylogenetic levels. Phylograms inferred from nuclear ribosomal DNA (FIGS. 2, 4) separated all three genera and indicated that Pilaira is distant from the other two genera. This conclusion agreed with O’Donnell et al. (2001), whose comprehensive study of the Mucorales SSU (including three isolates of species within Pilobolaceae) suggested that Pilaira should be removed from Pilobolaceae. The present study strengthened that suggestion because more isolates of each genus were included. The multiple isolates of Utharomyces and Pilobolus co-segregate into distinct clades inferred from the phylograms of both the SSU and ITS regions, strengthening the inference that these two genera are monophyletic. Even though it has been suggested that all mucoralean families but one be placed in the Mucoraceae (White et al. 2006), the results of our examination of the ITS and SSU DNA sequences and morphological characters such as trophocysts and subsporangial swellings demonstrated that Pilobolaceae should be retained as a separate family to include Pilobolus and Utharomyces. The phylogram inferred from these nuclear ribosomal DNA sequences resolved genus Pilobolus into two major clades, one of large spore-producing species, the second of small spore-producing species. The delimitation between the large and small sporeproducing species mirrors traditional morphological descriptions (Grove 1934, Zycha et al. 1969, Hu et al. 1989). Other studies report that sporangiospore size is one of the most stable morphological characteristics (Foos and Jeffries 1988). The present data show that this characteristic correlates with phylogenetic relationships. Polyphyly in Pilobolus.—In the phylograms inferred from nuclear ribosomal DNA the clade of small sporeproducing isolates (P. crystallinus, P. roridus and P. umbonatus) consists of two further clades, one of which contains P. crystallinus isolates IUE703 and NRBC 8561 and the other contains the remaining isolates of P. crystallinus as well as P. roridus and P. umbonatus. Separation of the isolates of P. crystallinus into clades suggests that the morphologically de-

scribed species comprises multiple phylogenetic species. Stajich et al. (2009) reported that strains that are similar in multiple morphological characteristics might be different phylogenetic species and that such polyphyly is not uncommon. However we cannot exclude the possibility that these isolates do in fact express yet undetected morphological characteristics that have not been reported in the literature. Data indicate a polyphyletic nature of morphological species of Pilobolus crystallinus and P. kleinii as supported by the bootstrap values in this molecular analysis. Wide variations in the quantitative features of the morphological characters used to identify species have led to misidentification or are the result of the polyphyletic nature of isolates that have similar morphological characteristics. Hu et al. (1989) proposed reducing Pilobolus umbonatus and P. roridus to a single species because they differed only in the shape of sporangia. However both SSU and ITS rDNA sequences of Pilobolus umbonatus differ greatly from those of P. roridus, placing them in different clades and thus supporting their recognition as different species. Likewise Hu et al. (1989) suggested reducing three species (P. crystallinus, P. kleinii and P. hyalosporus) to one species because they differed only in spore size and color. However spore size is supported in the molecular analysis as having great importance in differentiating species of Pilobolus, and this morphological evidence should not be disregarded by combining these three species into one. This study reveals that P. crystallinus and P. kleinii have different SSU and ITS rDNA sequences, in addition to different spore sizes, and should be retained as separate species. Because nuclear rDNA remains constant and characteristic it provides a reproducible and hence superior method with which to infer species of Pilobolus. Isolate IUE 566 was identified previously as both P. kleinii and P. crystallinus in different culture collections. Phylograms inferred from SSU and ITS sequences place IUE 566 in a clade with P. sphaerosporus, however sporangiospores size and shape are more characteristic of P. kleinii (FIG. 3B– D). This suggests either polyphyly in P. kleinii or the possibility of different morphological forms of P. sphaerosporus. Additional studies will be needed to clarify the relationship between these two species. Further work.—More effort will be required to reevaluate and redescribe existing isolates of Pilobolus available in culture collections. The traditional methods of identification and classification of Pilobolaceae based primarily on morphological characters are not sufficient to address the variability

FOOS ET AL.: PILOBOLACEAE associated with these organisms. Molecular phylogenetics can be used to determine not only the phylogenetic relationships within Pilobolaceae but also to identify Pilobolus isolates to species. Both morphological and molecular information can be used to distinguish between Utharomyces and Pilobolus. The molecular information presented here, in combination with spore size and shape, can be used to identify species of Pilobolus. In addition the rDNA sequences presented here will aid the characterization of new species of this genus. ACKNOWLEDGMENTS

This project was supported in part by an Indiana University sabbatical grant. We thank Kerry O’Donnell (National Center for Agricultural Utilization Research, Peoria, Illinois) and Lynne Sigler (University of Alberta Microfungus Collection and Herbarium) for providing isolates of fungal strains used in this work and Lawrence Washington (Indiana Molecular Biology Institute) for sequence analysis. KMF thanks Joan Henson for her hospitality and the use of her laboratory while developing the molecular techniques used in this study.

LITERATURE CITED

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