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A phylogenetic study of the genus Cookeina

     Harvard University Herbaria, 22 Divinity Avenue, Cambridge, Massachusetts 02138

Teresa Iturriaga

     Departamento de Biología de Organismos, Universidad Simón Bolívar, Apartado Postal 89000, Sartenejas, Baruta, Edo. Miranda, Venezuela

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Cookeina, with seven recognized species, is one of the commonly encountered genera of the Sarcoscyphaceae (Pezizales) in tropical and subtropical areas around the world. Morphologically the species are distinguished by combinations of several features including ascospore shape and surface relief, presence and origin of apothecial hairs and presence or absence of gelatinous material within the cortical layer of the excipular tissue. Color of the hymenium, attributed to carotenoid pigments, is particularly variable in some collections especially those referred to as C. speciosa. In this study phylogenetic analyses were carried out using rDNA ITS and rDNA LSU sequences. Forty-four collections were studied which included a broad sampling of color variants of C. speciosa from a field site in Venezuela. The genus was shown to be monophyletic with several well-supported lineages. These analyses generally support the established, morphologically distinguished taxa within a monophyletic genus Cookeina. Collections referred to as C. speciosa segregate within a clade in which hymenial color differences are associated with groups within the clade. Cookeina sinensis is sister to C. tricholoma but is distinct from it; C. indica fails to resolve with any of the major clades. The placement of C. insititia is ambiguous but it falls within Cookeina and thus is considered in the genus Cookeina rather than in a separate genus, Boedijnopeziza.

Key words: biogeography, ITS sequences, Pezizales, Sarcoscyphaceae

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Of the tropical and subtropical members of the order Pezizales few are as commonly encountered and collected as are species belonging to the genus Cookeina Kuntze (1891). Their brightly colored and relatively large apothecia assure that even the most casual observer will see them. The seven recognized morpho-species are defined on a series of distinctive and unambiguous macroscopic and microscopic characters related to occurrence and distribution of the hairs on the surface of the apothecium, spore shape and surface ornamentation, and presence or absence of gelatinous material in the tissue of excipulum. Species occur on fallen angiosperm branches, trunks and, occasionally, on durable fruits. Some taxa are widespread, such as C. colensoi (Berk.) Seaver, C. tricholoma (Mont.) Kuntze and C. speciosa (Fr. : Fr.) Dennis, but others are more restricted in distribution, such as, C. sinensis Z. Wang, C. venezuelae (Berk. & M. A. Curtis) Le Gal, C. insititia (Berk. & M. A. Curtis) Kuntze, and C. indica Pfister & R. Kaushal. Hymenial colors range from white to beige, yellow, orange, scarlet, and even to chocolate brown. Color variation is particularly pronounced among collections of C. speciosa. Arpin (1969) analyzed the pigments and reported carotenoids in two species, C. tricholoma and C. sulcipes (Berk.) Kuntze (=C. speciosa).

We initiated this study because we observed both distinct distributional patterns of species in this genus and variation in hymenial colors. The study focuses on the phylogenetic relationships among the taxa and their color variants. We obtained material of all taxa and we sampled broadly across the geographical range of taxa where we had appropriate material. A companion study will treat the genus monographically.

The family Sarcoscyphaceae, in which Cookeina has a central position, has a convoluted history, summaries of which may be found in Eckblad (1968), Korf (1970), Rifai (1968) and Harrington et al (1999). The Sarcoscyphaceae has been referred to the suborder Sarcoscyphineae (Rifai 1968). According to an analysis of the Sarcoscyphineae by Harrington et al (1999) it is a paraphyletic assemblage, but a clade containing Sarcoscypha (Fr.) Boud., Phillipsia Berk., Cookeina and related taxa is well supported and represents the Sarcoscyphaceae of most recent authors. In that study (Harrington et al 1999) Cookeina was shown to be sister to Microstoma Bernstein, a position that confirms Korf's (1972, 1973) view that these genera are closely related. Korf (1972) proposed a tribe, the Boedijnopezizeae, for Cookeina, Microstoma, and Boedijnopeziza S. Ito & S. Imai. These genera share features such as simultaneous maturation of ascospores within apothecia, a particular ascus construction in which the base of the ascus abruptly constricts to a thin supporting hypha and a dense network of anastomosing paraphyses. Microstoma is temperate in distribution. Boedijnopeziza, based on B. insititia, is known from Asia and is recognized as a distinct taxon (Rifai 1968, Korf 1972, 1973) or treated as a synonym of Cookeina (Le Gal 1953, Denison 1967, Pfister 1973, Pfister and Kaushal 1984). Korf (1983) suggested a relationship between the Boedijnopezizeae and Cyttaria Berk. Subsequent analysis of sequence data indicates that Cyttaria is only distantly related to the Pezizales (Landvik 1996).

Our goal in this study was to test the monophyly of the genus Cookeina and to evaluate the morpho-species as currently recognized, particularly with regard to biogeography and color variation. To accomplish our goal we used phylogenetic analyses of characters derived from nuclear encoded ribosomal DNA (rDNA).

	MATERIALS AND METHODS

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Material studied – Specimens of Cookeina used for DNA analysis were collected by the authors or obtained from collectors or herbaria (Table I). To distinguish specimens and avoid confusion with herbarium and field numbers, voucher numbers were assigned to collections. The ingroup consisted of 44 specimens of Cookeina, including 16 specimens of C. speciosa displaying distinct color variants. The outgroup was Microstoma floccosum.

DNA extract was used for polymerase chain reaction (PCR) of the internal transcribed spacers (ITS 1 and ITS 2) and the 5.8S region of the nuclear ribosomal DNA using the fungus-specific oligonucleotide primers ITS 4 and ITS 5 (White et al 1990). Four µL of PCR product was quantified on an ethidium bromide-stained 1.0% agarose gel, and the remaining PCR product was purified using Microcon 100 microconcentrator columns (Amicon Inc., Beverly, Massachusetts).

For dye-terminator cycle-sequencing involving the ITS region, ITS primers 2, 3, 4 and 5 were used; for the LSU region primers LR0R, LR3R, LR3 and LR5 were used. Sequencing reactions were run on an Applied Biosystems 377 automated DNA sequencer.

Analytical techniques – Sequences were edited and assembled using Sequencher 3.0 (GeneCodes, Ann Arbor, Michigan) and aligned manually in the data editor of PAUP 4.0d64 (Swofford 1991) with alignment gaps inserted to maximize aligned sites. Sequences have been deposited in GenBank (Table I) and the data matrix is available from TreeBASE as accession number SN1105.

Phylogenetic analyses were performed in PAUP 4.0d64 (Swofford 1991). After coding gaps in various fashions and finding basically similar results, we determined to treat gaps as missing data (gaps = missing coding); ambiguous regions in the alignment (characters 67–76, 79–93, 123–145, 160–182, 238–263, 266–275, 286–312, 530–545, 545–562, 687–716) were recoded as single characters (characters 782–791, respectively) using the methods described by LaGreca (1999), with each newly designated character given a weight equal to the number of characters from the corresponding ambiguous region. Using this system, the number of informative characters was increased from 246 to 256. Due to the size of the data set, we were limited to heuristic searches, which were performed in two parts. First, 100 heuristic searches were performed with random taxon addition and TBR branch swapping, with MAXTREES set to 200, keeping up to 2 trees per replicate. Second, all the shortest trees from the first part of the analysis were used as starting trees for complete TBR branch-swapping with MAXTREES set to 15 000. Relative robustness of individual clades was assessed by the bootstrap (Felsenstein 1985) using 100 heuristic searches, simple taxon addition sequences, TBR branch swapping and MAXTREES set to 1000.

Scanning electron microscopy (SEM), light microscopy (LM) and morphological studies – Ascospores of Cookeina collections were gathered by rehydrating a small piece of apothecium in several drops of water and then carefully separating the hymenial tissue from the excipular tissue. The hymenial tissue was macerated using a scalpel blade and further squashed with a pipette tip. An aliquot of this macerate was examined under LM. When free floating spores were observed, a drop of this spore suspension was pipetted onto a cover slip, dried, placed on a stub and sputter-coated with a gold-palladium alloy. Observations were made on an AMRAY model 1000 SEM.

Field studies – A detailed field study was undertaken in the Amazonian rainforest in Yutajé, Amazonas State, Venezuela. Cookeina species were collected in numbered plots on precisely designated and coded substrates. Thus, single collections could be traced precisely to a particular substrate and its neighbors on that substrate could be identified.

Color nomenclature – Colors were assigned in the field and correlated with Methuen (Kornerup and Wanscher 1978) color names and numbers. Methuen color notations are given in Table I.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Alignment – ITS1 and ITS2 were aligned along with the intervening 5.8S region and flanking partial sequences of 18S and 25S rDNA. All positions were generally able to be aligned within collections of the same species; alignment across species often required the addition of gaps. Because of the large data set this resulted in ten main areas of ambiguous alignment (as described in the Methods). The 5.8S region was almost identical across all taxa, and the aligned length of all sequences (including inserted gaps) was 781 bp.

Parsimony analyses – Under gap = missing data there were 256 informative characters yielding 15 000 equally parsimonious trees of 3083 steps (consistency index, CI = 0.960; retention index, RI = 0.980). Phylogenetic analysis identified four well-supported lineages of rDNA as measured by bootstrapping (Fig. 1). The monophyly of the C. speciosa and C. tricholoma lineages was strongly supported (for each, bootstrap = 100%) and the lineage consisting of C. colensoi and C. venezuelae was supported by a bootstrap value of 99%. Further bootstrap support of 100% and 93%, respectively, was obtained for both the C. colensoi and C. venezuelae clades, while the C. insititia clade was supported by a bootstrap value of 100%. The only specimen of C. indica, although falling within Cookeina, failed to clearly resolve with any of the greater clades.

View larger version (40K): [in this window] [in a new window]    FIG. 1. Strict consensus of 15 000 equally parsimonious trees (3083 steps) generated under gap = missing data. Numbers above the branches represent bootstrap support for 1000 replicates. Location codes are as follows: A—Australia, B—Borneo (Sabah), C—China, Co—Colombia, G—Guadeloupe, I—India, M—Mexico, NZ—New Zealand, PR—Puerto Rico, T—Thailand, V—Venezuela.

The C. tricholoma clade includes specimens from Sabah, China, Puerto Rico, Thailand and Venezuela. There is very little difference in ITS sequences within this clade despite the geographical spread of the collections (Fig. 2). Cookeina sinensis is basal in the C. tricholoma clade with 100% bootstrap support.

View larger version (29K): [in this window] [in a new window]    FIG. 2. Phylogram representing one of 1000 equally parsimonious trees for ITS sequences. Terminal taxa are individual collections (see Table I); branch lengths are according to scale. Geographical codes are as for Fig. 1.

Cookeina insititia also demonstrates considerable variation within the clade, especially considering that each of the four specimens analyzed were collected in China, three from Yunnan Province (Fig. 2).

SEM spore images – SEM images of spores from Cookeina (Fig. 3) aid and clarify descriptions derived from LM studies. Spores of several species are described as striate in LM studies but SEM reveals that the nature and arrangement of the striation varies. Spore ornamentation in C. speciosa, described as striate, consist of irregular anastomozing longitudinal ridges that appear somewhat wrinkled. Spores of C. tricholoma, also described as striate under LM, exhibit a more regular pattern of relatively straight and parallel longitudinal ridges. Cookeina indica, described as having fine longitudinal markings, has regular parallel longitudinal ridges that are wider than those in C. tricholoma. Cookeina venezuelae, typically described as having both longitudinal and transverse striations, can be more accurately described as having longitudinal ribs with fine transverse interconnecting ridges. Spores of both C. colensoi and C. insititia are uniformly smooth. Spores of C. sinensis were difficult to obtain because the material was scanty and questionably mature, but those we found were smooth.

View larger version (105K): [in this window] [in a new window]    FIG. 3. Ascospore ornamentation as seen by SEM. A. Cookeina speciosa. Face view showing anastomosing ridges (FH, Aldava 296). B. Cookeina indica. Face view showing 13–15 longitudinal ribbon-like ridges (HMAS, Z. Wang). C. Cookeina venezuelae. Profile view showing 6–7 longitudinal ribs with regular, fine transverse connecting ridges (FH 1107). D. Cookeina tricholoma. Face view showing 22–24 longitudinal ridges (OSC 67751). E. Cookeina colensoi. Face view (PDD 68628). F. Cookeina sinensis. Profile view (HMAS 14679). G. Cookeina insititia. Profile view, surface smooth (HMAS, Z. Wang). H. Microstoma floccosum. (FH, K. Griffith). Scale bar = 10 µm

	DISCUSSION

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General morphology – Phylogenetic analyses reveal several distinct rDNA lineages with resolution into clades that correlate with the morphological groups that have been recognized as species. The monophyletic group consisting of C. speciosa, C. tricholoma, and C. sinensis are hirsute, stipitate and lack a defined gelatinous layer in the excipulum at maturity. Spores are longitudinally ridged in the two former species and smooth in the latter. Cookeina venezuelae and C. colensoi, which also constitute a monophyletic group, have a well-defined excipular gel layer, are sessile or short stipitate and glabrous or with low scales or pustules. Spores are ornamented in C. venezuelae and smooth in C. colensoi. Cookeina indica, with ornamented spores, and C. insititia, with smooth spores, are intermediate regarding excipular features and did not clearly resolve in relation to the larger groupings.

Spore ornamentation has no clear correlation to any of the major lineages. Cookeina sinensis, for example, has smooth spores or slightly wrinkled spores (Wang 2001) yet resolves with taxa having striate spores, while C. venezuelae, with a unique spore ornamentation, as dissimilar to striate spores as they are to smooth spores, resolves with the smooth-spored C. colensoi. This lack of correlation contrasts with observations by Hansen et al (1999), who found that ITS lineages within Phillipsia, also a member of the Sarcoscyphaceae, were all supported by spore morphology.

Cookeina insititia is the only species with stipitate, hirsute apothecia and a gelatinous layer, but its relationship to one or the other of the main groups is uncertain. Likewise, C. indica, the only stipitate, glabrous species without a gelatinous layer, also fails to consistently resolve with one or the other of the greater monophyletic clades.

Color and regional variation in C. speciosa – Cookeina speciosa is noteworthy within the Pezizales for having a broad spectrum of apothecial colors. Our studies to date show no consistent anatomical differences among the color forms. On the other hand, rDNA analysis indicates collections in the same color ranges group together into two well-supported clades. Thus, one clade with a 65% bootstrap includes collections with darker colored apothecia, in the brown-mauve-dark coral (purplish) range; the second clade, with a 99% bootstrap, has lighter colored apothecia, in the yellow, orange, light coral, coral and white range. In Arpin's (1969) study he found no evidence for differences in the carotenoid content in two color variants of C. speciosa (as C. sulcipes) he studied.

In this study the northern region of South America represents the reference point from which variation can be assessed since a range of color forms were documented from there and incorporated in the data set. It is interesting therefore that the collection from Sabah groups with the more darkly pigmented specimens from Venezuela, while the collections from Thailand always group with the clade comprising the more lightly pigmented specimens from Venezuela. The sequence difference between the Sabah and Thailand specimens is considerable, given the geographical proximity of the two locations. This disparity cannot be accounted for by biogeographical features such as the Wallace line, which passes to the east of Borneo. The two regions are part of the same biogeographical zone, although associated with different forest types. Furthermore, field studies in Venezuela demonstrate a range of ITS and apothecial color differences present even within small areas and on the same pieces of downed wood.

Color as a reliable taxonomic character – Conventions in taxonomic mycology have had an important influence on species concepts within certain groups of fungi. Ascomycete systematists have largely discounted color variations in favor of reliance on microscopic characters. Although color variation was noted and used by some earlier mycologists to distinguish species of Cookeina, more recent authors have grouped the various color forms into more or less anatomically uniform morpho-species. Our data suggests that a closer look at these species complexes is required to understand the characters used in classification. We believe there are at least two taxa within the C. speciosa complex.

Resolution of the taxonomic status of C. sinensis – Cookeina sinensis was recognized on morphological grounds. It has smooth or slightly wrinkled spores but otherwise is morphologically similar to C. tricholoma. We examined both the sterile holotype and a fertile paratype of C. sinensis. Our morphological observations are still tentative because of the inadequacy of the available material. Even the paratype material is barely mature and it is possible that the smooth spores of C. sinensis could represent immature spores of C. tricholoma. Our studies indicate that the striations on ascospores of C. tricholoma form relatively late in the development of the ascospores. On molecular grounds C. sinensis falls outside and sister to the C. tricholoma group. The C. tricholoma collections from such disparate locations as Sabah, China, Puerto Rico, Thailand, and Venezuela, show short internal branch lengths (Fig. 2). While this lack of variation may somehow be unique to the biology of C. tricholoma, it also highlights the distinct taxonomic provenance of C. sinensis, which always resolves sister to the C. tricholoma collections (bootstrap = 100%). Furthermore, both specimens of C. sinensis and two specimens of C. tricholoma were collected in Yunnan Province of China, further weakening the argument that C. sinensis may represent either an immature collection of C. tricholoma or a regional variant of it. These phylogenetic results, along with the morphological difference that characterizes C. sinensis, provide evidence for its recognition as a distinct species. Wang (2001) reported C. sinensis from Taiwan and provided SEM photomicrographs of spores from a type collection and one from Taiwan.

Resolution of the taxonomic status of C. insititia – There has been debate over the taxonomic status of C. insititia. The spore shape, presence of a gel layer (known also in C. colensoi and C. venezuelae) and the form and origin of the hairs seem distinct in C. insititia. These features led Ito and Imai (1937) to erect a new genus, Boedijnopeziza. The genus was supported by some (Rifai 1968, Korf 1972, 1973) and disputed by others (Denison 1967, Eckblad 1968, Le Gal 1953, Pfister 1973, Pfister and Kaushal 1984). Molecular data supports the position of this species within the genus Cookeina, but because of lack of resolution in the strict consensus tree, its position in relationship to the other taxa remains problematic. Korf (1971) included C. colensoi in Boedijnopeziza based on the presence of gel in the excipular tissue of that species. Pfister (1973) recognized that C. colensoi and C. venezuelae shared this character. One could recognize Boedijnopeziza for C. insititia, C. colensoi, and C. venezuelae since the molecular data do not rule out such a position, but there remains little other than the somewhat ambiguous character of gel to unite them morphologically. In our opinion the choice to recognize Boedijnopeziza obscures the continuity of characters in this monophyletic group.

Distribution of C. colensoi – Denison (1967) reported that C. colensoi occurred chiefly south of the equator, with one collection from Venezuela and none from Central America. In our monographic studies we have determined that the Venezuelan collection represents a heretofore undescribed species. However, collections from Mexico, India, and China have come to light and have been included in this analysis. The species is best considered sub-tropical, with principle distribution in the southern hemisphere.

	ACKNOWLEDGMENTS

 
We thank the following people who provided specimens for our use in this study: Sharon Cantrell, Peter Johnston, Richard P. Korf, Thomas Læssøe, Zheng Wang, Wen-ying Zhuang and the curators of C, CUP, DAR HMAS, KMAS, PDD. We appreciate the comments given us by Meredith Blackwell and Scott LaGreca who read the manuscript in various versions. Scott LaGreca also assisted us with various technical aspects. We gratefully acknowledge the support provided by NSF DEB-9521944.

	FOOTNOTES

 
¹ Current address: Department of Botany, University of Tennessee, 326 Hesler Hall, Knoxville, Tennessee 37996

² Corresponding author, Email: dpfister{at}oeb.harvard.edu

Accepted for publication January 9, 2002.

	LITERATURE CITED

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Eckblad F-E., 1968 The genera of operculate discomycetes. A re-evaluation of their taxonomy, phylogeny and nomenclature. Nytt Mag Bot 15:1-191

Felsenstein J., 1985 Confidence limits on phylogenetics: an approach using the bootstrap. Evolution 39:783-791

Harrington FA, Pfister DH, Potter D, Donoghue MJ., 1999 Phylogenetic studies within the Pezizales. I. 18S rDNA sequence data and classification. Mycologia 91:41-50

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Korf RP., 1970 Nomenclatural notes. VII. Family and tribe names in the Sarcoscyphineae (Discomycetes) and a new taxonomic disposition of the genera. Taxon 19:782-786

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———. 1972 A synoptic key to the genera of the Pezizales. Mycologia 64:937-994

———. 1973 Discomycetes and Tuberales. In: Ainsworth GC, Sparrow FK, Sussman AS, eds. The fungi: an advanced treatise. Vol. 4A. New York: Academic Press. p 249–319

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White TJ, Bruns T, Lee S, Taylor JW., 1990 Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White T, eds. PCR protocols: a guide to methods and applications. San Diego: Academic Press. p 315–322

This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Weinstein, R. N. Articles by Iturriaga, T. Search for Related Content PubMed Articles by Weinstein, R. N. Articles by Iturriaga, T. Agricola Articles by Weinstein, R. N. Articles by Iturriaga, T.