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1 Department of Biology, Howard University, 415 College Street NW, Washington
D.C. 20059, U.S.A.
2 Agriculture and Agri-Food Canada/Agriculture et Agroalimentaire Canada,
Biodiversity (Mycology and Botany), 960 Carling Avenue, Ottawa, Ontario K1A
0C6, Canada
3 Department of Plant Pathology, Cornell University, 334 Plant Science Building,
Ithaca, New York 14853, U.S.A.
*
Correspondence: Priscila Chaverri
pchaverri{at}howard.edu
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Abstract |
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 TOP Abstract INTRODUCTIONTHE GENERA...MATERIALS AND METHODSRESULTSDISCUSSIONKEY TO GENERA TREATEDSYNOPTIC KEYSDICHOTOMOUS KEYSGENERA AND SPECIES DESCRIPTIONSEXCLUDED OR DOUBTFUL SPECIESReferences |
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(TEF
1-), and RNA polymerase II subunit 1 (RPB1) and analyses of multiple
morphological characters demonstrate that the three segregated genera can be
distinguished by the disarticulation of the ascospores and shape and size of
conidia. Moelleriella has filiform multi-septate ascospores that
disarticulate at the septa within the ascus and aschersonia-like anamorphs
with fusoid conidia. Hypocrella s. str. has filiform to long-fusiform
ascospores that do not disarticulate and Aschersonia s. str.
anamorphs with fusoid conidia. The new genus proposed here,
Samuelsia, has filiform to long-fusiform ascospores that do not
disarticulate and aschersonia-like anamorphs with small allantoid conidia. In
addition, the present study presents and discusses the evolution of species,
morphology, and ecology in Hypocrella, Moelleriella, and
Samuelsia based on multigene phylogenetic analyses. Taxonomic novelties: New genus: Samuelsia. New species: Hypocrella disciformis, H. hirsuta, Moelleriella basicystis, M. boliviensis, M. cornuta, M. evansii, M. madidiensis, M. umbospora, S. chalalensis, S. geonomis, S. intermedia, S. rufobrunnea, and S. sheikhii. New combinations: M. castanea, M. colliculosa, M. disjuncta, M. epiphylla, M. gaertneriana, M. globosa, M. guaranitica, M. javanica, M. libera, M. macrostroma, M. ochracea, M. palmae, M. phyllogena, M. rhombispora, M. sloaneae, M. turbinata, and M. zhongdongii.
Keywords multilocus phylogenetics / polyphasic taxonomy / species identification / species recognition
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INTRODUCTION |
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TOPAbstractINTRODUCTIONTHE GENERA...MATERIALS AND METHODSRESULTSDISCUSSIONKEY TO GENERA TREATEDSYNOPTIC KEYSDICHOTOMOUS KEYSGENERA AND SPECIES DESCRIPTIONSEXCLUDED OR DOUBTFUL SPECIESReferences |
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Molecular and morphological characters support the recognition of three well-defined genera within the broad concept of Hypocrella: Hypocrella s. str., Moelleriella Bres., and Samuelsia gen. nov. (Clavicipitaceae, Hypocreales, Hypocreomycetidae, Sordariomycetes, Pezizomycotina, Ascomycota). These new genera and their anamorphs are presented here. Hypocrella s. str. has an Aschersonia anamorph. The anamorphs of Moelleriella and Samuelsia are aschersonia-like and thus hereafter will be denoted in quotes (e.g. "Aschersonia") to avoid confusion.
Until 1897, Hypocrella s. l. species were described as phyllogenous and thought to be either parasitic or superficial colonisers of living leaves (Evans & Hywel-Jones 1990). Their parasitic association with scale insects and whiteflies was not recognised until Webber (1897) concluded that several Moelleriella species (then identified as "Aschersonia" aleyrodis Webber) were important factors in the biological control of whitefly pests in citrus plantations. Indeed, the first biocontrol applications in the U.S.A. were done with "A." aleyrodis (teleom. Moelleriella libera) to control citrus whitefly (Dialeurodes citri Ashmead) in Florida (Berger 1921). Since then, the biocontrol potential and ubiquity of Hypocrella s. l. has been widely recognised (Parkin 1906, Morrill & Back 1912, Petch 1921, Fawcett 1936, Ferron 1978, Barua 1983, Brady 1984, Ramakers & Samson 1984, Fransen 1987, Rombach & Gillespie 1988, Gerling 1992, Osborne & Landa 1992, Meekes et al. 1994, Samson 1995, Meekes et al. 1996, Lourencao et al. 1999, Meekes et al. 2000, Faria & Wraight 2001, Meekes 2001).
Despite the potential of these species in insect biocontrol, few taxonomic treatments have been written after Petch's (1921) monograph of Hypocrella s. l. Approximately 115 names in Hypocrella and 79 names in Aschersonia have been validly published; however, only about 50 and 44 species, respectively, are currently accepted (Petch 1921, Dingley 1954, Mains 1959b, a, Hywel-Jones & Evans 1993). The present taxonomic treatment deals with Hypocrella s. str. (anamorphs Aschersonia s. str.) and its newly segregated sister genera Moelleriella and Samuelsia that are encountered in the Neotropics. Hundreds of freshly collected and herbarium specimens, including types were examined, and multigene phylogenetic analyses were conducted. Even though this is a comprehensive study, fungal biodiversity surveys in other poorly explored regions will probably reveal more undescribed species.
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THE GENERA HYPOCRELLA/ASCHERSONIA, MOELLERIELLA, AND SAMUELSIA |
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TOPAbstractINTRODUCTIONTHE GENERA...MATERIALS AND METHODSRESULTSDISCUSSIONKEY TO GENERA TREATEDSYNOPTIC KEYSDICHOTOMOUS KEYSGENERA AND SPECIES DESCRIPTIONSEXCLUDED OR DOUBTFUL SPECIESReferences |
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The genus Hypocrella was erected by Saccardo (1878) to accommodate four species previously assigned to Hypocrea Fr. (Hypocreaceae, Hypocreales): Hypocrea atramentosa Berk. & M.A. Curtis (= Myriogenospora atramentosa (Berk. & M.A. Curtis) Diehl); Hypocrea discoidea Berk. & Broome; Hypocrea semiamplexa Berk. (a Balansia species according to Petch 1921); and Hypocrea bambusae Berk. & Broome (= Balansia bambusae (Berk. & Broome) Petch). Only Hypocrella discoidea (Berk. & Broome) Sacc. (type of the genus) remains in Hypocrella. Moelleriella was erected in 1896 to accommodate Moelleriella sulphurea Bres. (= M. phyllogena). In Bresadola's original diagnosis, M. sulphurea has a pulvinate stroma and filiform ascospores that disarticulate into numerous cylindrical part-spores.
The genus Hypocrella was originally distinguished from Hypocrea by the formation of eight, filiform ascospores in each ascus (Saccardo 1878). Petch (1921) reported that the filiform ascospores in H. discoidea disarticulated at the septa while still in the asci and speculated that if whole ascospores were seen in asci, it was an indication of immaturity. Later, Petch (1939) described a new genus, Stereocrea H. Sydow & P. Sydow, where he placed species with non-disarticulating ascospores. At that time, Petch did not realise that H. discoidea also had non-disarticulating ascospores. Later, Mains (1959b) concluded that the presence of non-disarticulating ascospores in H. aurantiaca (Petch) Mains (= Stereocrea aurantiaca) was not enough evidence to separate it from Hypocrella. Until now, it had been accepted that Hypocrella species include those with disarticulating and non-disarticulating ascospores (Petch 1939, Mains 1959a, b, Hywel-Jones & Evans 1993). However, evidence presented in this study supports speculations by Hywel-Jones and Evans (1993) and Petch (1939) that species with disarticulating ascospores form a monophyletic group that should be segregated from Hypocrella. In the present monograph, species with disarticulating ascospores are placed in Moelleriella.
Anamorphs of Hypocrella have been classified in the genus Aschersonia, which was erected by Montagne (1848) based on A. tahitensis Mont. Aschersonia s. str. is characterised by pycnidium-like conidiomata, phialides, paraphyses, and unicellular, fusiform, hyaline conidia that are brightly coloured in mass and produced in copious slime. Moelleriella, Hypocrella and Samuelsia species may or may not have paraphyses. The presence of paraphyses (sterile, hypha-like elements in the hymenium) has sometimes been used as a character to distinguish Aschersonia subgenera. Moelleriella and Samuelsia also have aschersonia-like anamorphs that are similar to Aschersonia s. str. but can be distinguished by conidial size and shape. Hypocrella s. str. has fusiform conidia that are larger than those of Moelleriella and Samuelsia. Samuelsia has allantoid and smaller conidia. The anamorphic states of Hypocrella, Moelleriella, and Samuelsia are more commonly collected than the teleomorphs, and are rarely associated with the teleomorph in the same stroma.
As discussed in Chaverri et al. (2005a), within the Clavicipitaceae, pycnidial to acervular anamorphic forms have been assigned to one of three anamorph genera: Aschersonia, Ephelis Fr., or Sphacelia Lév. These types of anamorphs are known only for the plant-associated teleomorph genera Atkinsonella Diehl, Balansia Speg., Claviceps Tul., Epichloë (Fr.) Tul. & C. Tul., Myriogenospora G.F. Atk. and Neoclaviceps J.F. White et al., and the scale-insect and whitefly parasites Hypocrella, Moelleriella, Samuelsia, and Regiocrella. Some studies have shown evidence of a single evolutionary origin of the pycnidial-acervular morphology in the Clavicipitaceae (Kuldau et al. 1997, Sullivan et al. 2001, Chaverri et al. 2005a). Regiocrella was recently described and although similar to Hypocrella, Moelleriella, and Samuelsia in its pycnidial-acervular conidiomata, brightly coloured ascomata, and its parasitism on scale insects, Regiocrella differs from those three genera by its short-fusiform and unicellular ascospores (Chaverri et al. 2005a). Within the group of genera with pycnidial-acervular conidiomata, Hypocrella, Moelleriella, and Samuelsia are easily distinguished by the shapes of their conidia and ascospores.
The first suggestion that Aschersonia was the anamorph of Hypocrella was made by Massee (1896) in an account of Aschersonia oxyspora Berk. (= Moelleriella phyllogena = H. phyllogena (Mont.) Petch). Later, Möller (1901) reported on the occurrence of both the perithecial and pycnidial states in the same stromata of H. cavernosa A. Möller (= M. cavernosa = M. palmae Berk. & M.A. Curtis), and identified the pycnidial state as aschersonia-like. Although it is not common to find both states in the same stroma, several species of Hypocrella, Moelleriella, and Samuelsia possess this characteristic (e.g. M. libera, M. mollii, M. ochracea, M. reineckiana, M. sloaneae, M. turbinata, H. disciformis, H. discoidea, H. viridans, and S. rufobrunnea, among others). It is now widely accepted that Aschersonia is the anamorph of Hypocrella. However, the whole life cycles of many of the 40-50 species of Hypocrella, Moelleriella, and Samuelsia are not yet known. Only about 15 species of Aschersonia s. l. have been linked to their teleomorphs (Petch 1921, Dingley 1954, Mains 1959b, a, Hywel-Jones & Evans 1993). In the present study, previously unknown life cycles of Hypocrella, Moelleriella, and Samuelsia are described and illustrated (see Table 1 for teleomorph-anamorph connections).
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Taxonomic Background: Subdividing Hypocrella/Aschersonia s. l.
Few studies have comprehensively dealt with the taxonomy of
Hypocrella and related genera or have included more than a few
species (Petch 1921, Mains
1959a,
b,
Liu et al. 2006). The
first taxonomic treatment of the genus was made by Parkin
(1906) who considered
extensive collections from Sri Lanka. The seminal monograph of Petch
(1921) treated 57 species of
Hypocrella, including anamorphs and teleomorphs from the New and Old
Worlds. Mains (1959a,
1959b) dealt with 24 species
of Hypocrella and 15 species of Aschersonia.
Petch (1921) proposed Hypocrella subgenus Fleischeria for species of Hypocrella in which the Aschersonia state lacks paraphyses (and Aschersonia subg. Leprieuria for the corresponding anamorphs). He considered that most of these species attack scale insects (Coccidae; Lecaniidae according to Petch 1921). In that same work, Petch proposed Hypocrella subg. Hypocrella (as "Euhypocrella" see Art. 21 ICBN) for species on Aleyrodidae and with paraphyses in the conidiomata of their Aschersonia subg. Aschersonia (as "Euaschersonia" see Art. 21 ICBN) anamorphs. Recent evidence reveals that, although sometimes diagnostic for individual species, the presence of paraphyses in the conidioma is not phylogenetically informative (Liu & Hodge 2005, Liu et al. 2006). Dingley (1954) also questioned this division since "A." duplex Berk., with paraphyses in the conidioma, is consistently associated with Coccidae. In addition, other studies showed that cultures of Aschersonia may lack paraphyses whereas these were present in the conidioma on the host, further indicating that the presence of paraphyses is not a stable taxonomic character (Hywel-Jones 1993, Hywel-Jones & Evans 1993, Meekes 2001).
Petch (1921) also discussed the possibility of splitting Hypocrella into groups characterised by the form of the stroma. However, he mentioned that these forms "...grade into one another to such an extent that this character cannot be relied on." In the original diagnosis of the genus, Fleischeria was distinguished from Hypocrella by its harder stroma (Penzig & Saccardo 1901). Petch (1921) synonymised the two genera because species of Hypocrella can vary in hardness, from hard in H. schizostachyi Henn. (= M. schizostachyi) to soft in H. convexa Racib. (= M. convexa). Chaverri et al. (2005b) demonstrated the presence of three natural groups (i.e. clades) in Hypocrella s. l. based on DNA sequence data that correlated with stromatal morphology. The Effuse group has flat, effuse stromata of loose hyphal tissue, broad hypothalli, and whitish colouration (e.g. M. evansii, M. libera, M. madidiensis, M. ochracea, M. raciborskii, M. rhombispora, and M. zhongdongii), except pale yellow to orange in the M. basicystis species complex; the Globose group includes species that have globose stromata that are generally darker in colour (yellow to brownish), large, compact tissue, hard or coriaceous, moderately to strongly tuberculate, and without hypothalli (e.g. M. africana, M. boliviensis, M. cornuta, M. epiphylla, M. gaertneriana, M. insperata, M. macrostroma, M. turbinata, and M. schizostachyi); and the Pulvinate group (now Hypocrella and Samuelsia) comprises species that have pulvinate or cushion-like stromata, somewhat compact and flattened, yellowish or green, sometimes brownish, with or without hypothalli, generally changing colour in 3 % KOH (e.g. H. aurantiaca, H. disciformis, H. viridans, H. discoidea, H. hirsuta, S. geonomis, S. chalalensis, S. rufobrunnea, and S. sheikhii). The groups defined by Chaverri et al. (2005b) correlate roughly with some of the groupings that Petch (1921) discussed. Although there are distinct phenotypic characteristics that distinguish each of the three major groups defined in Chaverri et al. (2005b), a formal generic or subgeneric revision of the taxonomy to distinguish the Effuse, Globose, and Pulvinate groups was not proposed.
Geographical Distribution
Hypocrella, Moelleriella, and Samuelsia are mainly
distributed in the Tropics, with a few species found in the Subtropics
(e.g.M. colliculosa, M. duplex, M. guaranitica, S. intermedia, H.
aurantiaca, and H. citrina)
(Table 1). This pattern of
distribution and evolutionary dynamics have been observed in many other
organisms where taxa preferentially originate in the Tropics and expand toward
the poles without losing their tropical presence
(Jablonski et al.
2006). This might explain the higher biodiversity of fungi in the
tropics compared to that in temperate or arctic regions. Therefore, a tropical
diversity crisis would have profound evolutionary effects at all
latitudes.
In Hypocrella and Moelleriella but not Samuelsia, taxa with a disjunct distribution are apparent (Lee et al. 1996, Taylor et al. 1999, Wen 1999, Wu et al. 2000). A disjunct distribution is one in which two closely related taxa (i.e. morphologically similar) are widely separated geographically. In Hypocrella and Moelleriella, an Old World (OW)/New World (NW) disjunction is sometimes observed (Petch 1921, Evans 1982, Evans & Hywel-Jones 1990). Disjunctions have been observed in M. palmae (NW) vs. M. sclerotioides Höhn. (OW); M. epiphylla (NW) vs. M. reineckiana (OW); M. libera (NW) vs. M. raciborskii (OW); M. ochracea (NW) vs. M. mollii (OW); M. macrostroma (NW) vs. M. africana (OW); M. castanea Petch (NW) vs. M. palmicola Henn. (OW); and M. gaertneriana (NW) vs. M. schizostachyi (OW) (Petch 1921, Evans 1982, Evans & Hywel-Jones 1990, Chaverri et al. 2005b). It is possible that disjunct geographical distributions are common in Hypocrella and Moelleriella, but, because this monograph focuses on New World species, this issue is beyond the scope of the study.
Individual species of Hypocrella, Samuelsia, and Moelleriella may be distributed over a large region or may be endemic to smaller areas. The presumably short dispersal distances of spores, the distributions limited to the Tropics, and the relatively low intraspecific genetic variability suggests the majority of the species form geographically conserved populations (Obornik et al. 2000). For example, in the New World Tropics, M. epiphylla, M. libera, M. ochracea, M. rhombispora, M. turbinata, and M. zhongdongii are widespread and ubiquitous. In contrast, M. gaertneriana and M. cornuta have been found only in the Amazon basin (i.e. Brazil, French Guiana, Venezuela) and are rare. Moelleriella evansii and S. chalalensis, S. geonomis, and S. rufobrunnea have been found only in Ecuador, Bolivia, and Peru, respectively. The phenomenon of geographically restricted species is also observed in the M. basicystis species complex: M. basicystis s. str., M. phyllogena, M. disjuncta, and M. umbospora. The present monograph demonstrates that M. basicystis has been found in Panama and Costa Rica (probably southern Central America); M. phyllogena in Panama, Brazil, Ecuador, Bolivia, and Peru (probably Panama and Amazon basin); M. umbospora in Mexico, Honduras, Guatemala (probably northern Central America); and M. disjuncta in Panama and Guyana.
Increased collecting in poorly explored regions will very likely discover new and rare species of Hypocrella, Samuelsia, and Moelleriella. However, many species are apparently restricted to undisturbed old growth tropical forests. The increased destruction of these sensitive habitats due to deforestation, forest fires, urban sprawl, and other factors, will result in the disappearance of fungal species, some with potential beneficial value to human societies (e.g. as biocontrols and sources of novel metabolites) (Evans 1982, Samson & Evans 1985, Evans 1988, Samson 1995, Chaverri & Vílchez 2006).
Habitat
Microclimate
Although Hypocrella, Samuelsia, and Moelleriella are
widespread throughout the Tropics, they do not occur in all types of climates
and habitats within the Tropics. Few publications report on the climate or
habitat characteristics that Hypocrella or Moelleriella
species prefer, and there is no published information on Samuelsia
species. Based on studies done for other fungi, ultraviolet light and direct
solar radiation may be one important factor affecting the survival of
entomopathogenic fungal spores (Fargues
et al. 1996, Moore
et al. 1996), as well as scale insects and whiteflies.
This explains in part why species of Hypocrella, Samuelsia, and
Moelleriella are found mostly on the abaxial surface of leaves, where
the insect and the fungus are protected from direct solar radiation. For
example, during the survey of Hypocrella, Samuelsia, and
Moelleriella done for the present study, no specimens were found in
open highly sun-exposed areas lacking a tree/shrub canopy.
Temperature may also affect survival of Hypocrella, Samuelsia, and Moelleriella. Some studies have shown that temperature may affect conidial germination and nymphal mortality by M. libera (anam. "A." aleyrodis) (Fransen et al. 1987, Fransen 1995). In contrast, Meekes et al. (2000) considered that temperature did not greatly affect infection ability by M. libera, but found a slightly higher conidial germination rate at 25 °C compared to 20 °C. In the present study, it was observed that habitats with a combination of suboptimum temperatures (ca. 28-38 °C or below 18 °C) and extreme continuous relative humidity conditions (e.g. dry forests, very humid forests) did not produce many fungal collections. For example, six weeks of surveying a lowland very wet tropical forest in Costa Rica yielded only eight species of Hypocrella s. l. (Chaverri & Vilchez 2006). Similarly, Hywel-Jones and Evans (1993) in Thailand, did not find specimens of Hypocrella s. l. during the driest months of the year (February, March, and April). However, high relative humidity (ca. 80 %) may be necessary for increased conidial germination and whitefly mortality by M. libera and M. raciborskii (Meekes 2001). The environment immediately surrounding the conidia may also influence their survival. Chemicals on the leaf surface may influence the viability of spores, but also they can have an effect on the pest and its susceptibility to pathogens (Hare & Andreadis 1983, Cooke & Rayner 1984, Ramoska & Todd 1985).
Host specificity, co-evolution, and adaptation
Species of Hypocrella, Samuelsia, and Moelleriella are
scale-insect and whitefly pathogens. The insect host is almost always
completely consumed by the fungus before the stroma and fruiting structures
become evident (Evans 1988,
Evans & Hywel-Jones 1990,
Meekes et al. 1994),
at which point it is almost impossible to identify the insect. Therefore,
there is scarce information about host specificity in Hypocrella,
Samuelsia, or Moelleriella. Petch
(1921) stated that species of
Hypocrella could parasitise either whiteflies (Aleyrodidae)
or scale insects (Coccidae and Lecaniidae). In some cases,
it may be possible to hypothesise the identity of the insect host based on
neighboring individuals that are not completely consumed by the fungus or not
infected. However, this approach can be misleading, because in many cases
several species of scale insects or whiteflies are found on the same leaf
(Petch 1921). Some species
such as M. libera/"A." aleyrodis, M.
raciborskii/"A." placenta, and M.
ochracea/"A." andropogonis appear to be generalists and have
been found to infect at least five scale-insect and whitefly species
(Petch 1921,
Meekes et al. 2000,
Meekes 2001,
Meekes et al. 2002).
In most species of Hypocrella s. l. the degree of host specificity is
completely unknown.
Characteristics of the plant upon which the host insect is feeding may also be important. A single strain of M. libera showed differences in persistence as a consequence of chemical and/or morphological differences between plants (Hare & Andreadis 1983, Cooke & Rayner 1984, Ramoska & Todd 1985, Meekes et al. 2000). The co-evolution of fungi and their insect hosts may reflect co-evolution between the insects and their plant hosts. In tropical forests, the spatial separation between conspecific plant individuals may have given rise to spatially separated insect populations that, in turn, coevolved with their fungal parasites (Evans 1988). Successful horizontal dispersal of the pathogens between coccid and whitefly colonies would have decreased proportionally with increasing adaptation and restriction of their hosts to certain trees (Evans 1988).
Epizootics caused by Hypocrella, Samuelsia, and Moelleriella on coccids and whiteflies in the Tropics are so prominent that several authors have wondered how these insects survived (Petch 1925, Evans 1974, 1982, 1988). As mentioned before, dispersal of the fungus probably played an important role in the co-evolution with its insect host, and the co-evolution of the insect with its plant host. The anamorphic forms of Hypocrella, Samuelsia, and Moelleriella produce slimy masses of conidia that are well adapted for short-distance, water-borne movement over leaf surfaces (Parkin 1906, Chaverri & Samuels 2003, Hodge 2003). The mucilage that covers the conidia is high in sugar content making the conidia more hygroscopic (Meekes 2001) and attractive to insects. This mode of short-distance dispersal is apparently so efficient that once a colony of insects becomes infected by the fungus, it is difficult to find healthy individuals (Evans 1988).
Evans (1988) speculated about insect adaptation to the threat from entomopathogenic fungi. The short-distance dispersal of conidia that contributes to epizootics may select for discontinuous patterns of insect distribution. On the other hand, the teleomorphic forms of Hypocrella, Samuelsia, and Moelleriella are less commonly encountered and have dry, discharged ascospores that are probably wind-dispersed and so better adapted for medium to long distance dispersal (Evans 1988, 1989). However, the above hypothesis could be refuted because in related genera, such as Sphacelia, Ephelis and Neotyphodium, the discharge of conidia in slime appears to aid dispersal by insects (Loveless 1964, Mower et al. 1973, Mower & Hancock 1975, Samways 1983, Butler et al. 2001, Hodge 2003).
Scale insects and whiteflies often secrete sticky honeydew that is attractive to wasps and ants, so it is also possible that Hypocrella, Samuelsia, and Moelleriella evolved to produce slimy conidia that adhere to non-host insect vectors. Infected coccid or whitefly alates may further transport the fungus, as shown for some aphid-Entomophthorales interactions (Wilding & Perry 1980, Evans 1989). Insect vectors and infected alates could disperse the spores across long distances; water, such as rain splash and run-off, could disperse across short distances (Chaverri et al. 2005a).
Plant-insect-fungus specificity is suspected but not well known in Hypocrella, Samuelsia, and Moelleriella, and further experimental evidence will be important in developing these fungi as biological controls. Based on observations made for the present study, the majority of the specimens were found on shrubs, trees, palms, and Musaceae leaves, with a few species (e.g. M. libera, M. ochracea, H. disciformis, and H. viridans) found on small herbaceous plants. The plant-specificity of the host insects may be important in determining where the fungi are found; scale insects and whiteflies are themselves poorly understood, with the exception of a handful of species that are crop pests. Samuelsia geonomis, S. chalalensis, S. rufobrunnea, M. gaertneriana and M. schizostachyi have been found only on monocotyledonous plants, such as bamboo culms and palm leaves; and the related species M. africana and M. macrostroma have been found only on stems of dicotyledonous plants (Hywel-Jones & Samuels 1998, Chaverri et al. 2005b). Although most species of Hypocrella, Samuelsia, and Moelleriella are found on leaves, other species such as M. epiphylla and M. turbinata are found both on leaves and stems (Petch 1921, Mains 1959a, b). A few other species that are more widespread can be found both on monocot and dicot leaves (e.g. M. basicystis, M. libera, M. ochracea, and M. phyllogena). The great majority of the species of Hypocrella, Samuelsia, and Moelleriella are found on leaves, with a larger portion occurring on the abaxial surface of leaves and a smaller percentage on the adaxial surface (e.g. M. epiphylla, M. turbinata, M. reineckiana) (Petch 1921, Hywel-Jones 1998). Whitefly and scale-insect nymphs are mainly present on the abaxial surface of leaves.
Nutrition
The mechanisms by which Hypocrella, Samuelsia, and
Moelleriella species obtain enough nutrients from the host to support
the relatively large size of the stromata are not well studied. In
Moelleriella, several species have especially large stromata: M.
gaertneriana, M. africana, M. schizostachyi, and M. macrostroma.
Other genera in the Clavicipitaceae, including Ascopolyporus
A. Möller, Dussiella Pat. and Hyperdermium, also
parasitise scale insects and have relatively large stromata. In these genera,
the stromatal mass greatly exceeds that of the scale-insect host. Sullivan
et al. (2000)
hypothesised that the large size of the stromata results from a kind of
secondary plant parasitism. Once the fungus has consumed the scale-insect
body, the fungus may continue to access plant nutrients through the insect's
stylet. Another hypothesis suggests that the mechanism of nutrient acquisition
in fungal species parasitic on scale insects and whiteflies is through the
living insect that forms a bridge between the fungus and the plant
(Couch 1938,
Hywel-Jones & Samuels
1998). Koroch et al.
(2006) studied host nutrient
adaptation in Balansia henningsiana (a plant pathogen) and M.
phyllogena (a scale-insect pathogen). They observed that both fungi
exhibit a restricted range of similar nutrient sources that may support growth
and that those nutrients (such as sucrose) are most likely from plant sources.
This same study suggests that the scale-insect pathogen M. phyllogena
obtains some of its nutrients from the host plant rather than exclusively from
the insect. In terms of its nutrition, M. phyllogena is more similar
to the plant biotrophs than to entomopathogenic fungi in the same family
(Koroch et al. 2006).
Its close evolutionary relationship with plant pathogens has been supported by
previous studies (Bischoff et al.
2004, Chaverri et al.
2005a).
Hypocrella, Samuelsia, and Moelleriella species can usually be grown in culture on most standard laboratory media. Growth rates are relatively slow, but most species will produce conidia in culture. Typical stromata are not formed in culture, and sexual fruiting bodies have not been observed.
Life cycle and epidemiology
Host and conidia may meet in two different ways: (1) direct contact, when
conidia are released/introduced upon the insect host (dispersed by water, air,
or other insects), or (2) indirect contact, when hatching or moulting nymphs
(larvae) settle on or near conidia already present on the leaf surface
(Meekes 2001). The sticky
mucilage that covers the conidia permits them to adhere to hydrophobic
surfaces, such as the insect cuticle in the presence of water
(St.-Leger 1991,
Meekes 2001). The whitefly or
scale insect is infected when germinating spores penetrate the insect cuticle.
Germination and appressorium formation in several aschersonia-like species
does not seem to be affected by the specific binding site on the insect nor by
a specific instar (Meekes
2001). The ideal temperature for germination seems to be
ca. 25 °C, but as low as ca. 20 °C and as high as
ca. 30 °C; germination and germ-tube length is severely limited
at 15 °C and 35 °C (Ibrahim et
al. 1993). Studies also show that exposure of conidia to
extended periods of time at 25 °C or higher reduce their viability after
ca. 15 d (Fransen
1995, Meekes et al.
2000, Meekes et al.
2002). The ability of Hypocrella, Samuelsia, and
Moelleriella conidia to remain viable in a potential habitat of the
host insect may be influenced by temperature, relative humidity, solar
radiation, characteristics of the leaf surface (chemical and/or morphological
differences between the plants), canopy characteristics, and the presence of
other microorganisms on the leaf. Conidial germination capacity can remain
high for at least one month. With respect to ascospores, there is no known
information on the factors that influence their germination and viability.
Conidia of most species of Hypocrella, Samuelsia, and
Moelleriella germinate in favorable conditions after 24-48 h
(Ibrahim et al. 1993,
Fransen 1995,
Meekes et al. 2000,
Meekes et al.
2002).
Under greenhouse conditions, most infections occur during the night following the release of conidia. In M. libera, the first sign of infection is a discolouration of the first instar larvae 4-10(-14) d after inoculation (Samson & Rombach 1985, Meekes et al. 2000, Meekes et al. 2002). The rate of infection declines with increased age of the insect; the fourth instar, prepupae, and pupae are less susceptible. Eggs are not infected. After infection, the fungus proliferates inside the host by first forming hyphal bodies, a yeast-like stage. Once the mycelium has fully colonised the body cavity, it emerges from the insect and forms a fringe around the insect's body, apparently adhering it to the plant host.
Sporulation occurs early in the infection process, soon after the hyphae rupture the dorsal cuticle and produce mat-like pustules of white mycelia on the host surface (Samson & Rombach 1985, Meekes et al. 2000, Meekes et al. 2002). Pycnidia appear to form first. This is supported by observations of Hywel-Jones and Evans (1993), who found the anamorph in the first months of the wet season and the teleomorph in the last months of the wet season, right before the dry season started. In a few cases, pycnidia and perithecia may be present in the same stroma at the time of collection. Many of the teleomorph collections for the present monograph were made from fallen leaves that were on the forest floor.
In culture, conidia and ascospores of several species of Hypocrella and Moelleriella (H. discoidea, M. epiphylla, M. turbinata) germinate to produce long and slender conidiogenous cells, and then secondary conidia (capilliconidia) (Hywel-Jones & Evans 1993, Evans 1994, Meekes 2001). This may be a mechanism to ensure secondary dispersal if the primary spores (i.e. ascospores or conidia) do not reach the target insect. This phenomenon has been observed in some entomophthoralean fungi (King & Humber 1981, Keller 1991, Hywel-Jones & Evans 1993). It is thought that these capilliconidia are formed in response to the absence of a suitable host (King & Humber 1981, Humber 1984, Evans 1994) or as a mechanism to increase the chances of transmission to mobile host insects (Glare et al. 1985b, a). Other types of anamorphs (i.e. synnematous or mononematous synanamorphs) have also been observed in Hypocrella and Moelleriella: hirsutella-like in M. insperata Rombach et al. (Liu et al. 2005), M. turbinata, M. schizostachyi (Hywel-Jones & Samuels 1998), and Hypocrella hirsuta. This type of synanamorph is usually produced in culture at an early stage in the development of the stroma, and is followed later by the aschersonia-like form (Liu et al. 2005).
Species of Hypocrella, Samuelsia, and Moelleriella are not known to produce resting spores, chlamydospores, or other structures that persist during unfavorable environmental conditions. Carotenoids or similar pigments in their stromata and conidia may contribute to long-term survival—most species are brightly coloured, mostly orange, due to these pigments (Eijk et al. 1986). Pigments may enhance the ability of the spores to withstand short periods of exposure to solar radiation.
Although sexual fruiting bodies of Hypocrella, Samuelsia, and Moelleriella are found in nature, nothing is known of the stimuli or requirements for sexual reproduction. Based on observations made for the present study, no perithecia form in culture. It is possible that the genera are heterothallic or the environmental conditions are not conducive for the formation of perithecia and ascospores. This is also the case for most other clavicipitaceous fungi, among which the development of teleomorphs in culture is rare. Only a few insect pathogens have been reported to fruit in culture after artificial manipulations. For example, Cordyceps militaris (L.) Link, Torrubiella spp., and Romanoa Thirum. have been observed to produce perithecia in semisynthetic media (Thirumalachar 1954, Basith & Madelin 1968, Hodge 2003). Sung (1996) induced the formation of stromata and perithecia in several Cordyceps species using media composed of sterilised brown rice with chopped silkworm pupae.
Secondary metabolites produced by Hypocrella/Aschersonia and Moelleriella
The death of insects invaded by ascomycetous fungi is thought to be caused
by toxins released by the fungus (Roberts
1981, Evans 1988).
Evans (1988) hypothesised that
toxins probably build up in the haemocoel of the insect as the yeast-like
parasitic cells colonise the circulatory system. There is little information
regarding secondary metabolites or toxins produced by Hypocrella,
Samuelsia, and Moelleriella, mostly because of the lack of
available cultures for studies (Isaka
et al. 2003). Watts et al.
(2003) demonstrated that the
anthraquinone dimers, rugulosin and skyrin, extracted from H.
discoidea were cytotoxic to some insect cells (i.e. Spodoptera
frugiperda). Watts et al.
(2003) reported that most of
the isolates positive against insect cells were from
"...Hypocrella with whole ascospores...". Destruxins
(cyclohexadepsipeptides), which exhibit insecticidal, phytotoxic, antiviral,
cytotoxic, and immunodepressance activities, have been found in various
entomopathogenic fungi, including Moelleriella and
Hypocrella (Krasnoff et
al. 1996). These few studies support the hypothesis that
secondary metabolites may be involved in insect pathogenicity.
The compounds hypocrellin A and B have been erroneously linked to
Hypocrella (Zhang et
al. 1989, Hudson et
al. 1994, Diwu
1995, Zhang et al.
1998, Fei et al.
2006). This compound is only known from
"Hypocrella" bambusae, which is actually a
Balansia (Petch
1921). A few other secondary metabolites that have been identified
but not linked to a function in vivo are triterpenes 3β,
15, 22-trihydroxyhopane from M. libera
(Eijk et al. 1986)
and an analog from M. tubulata
(Boonphong et al.
2001). These compounds exhibit activity against Mycobacterium
tuberculosis. Zeorin (6, 22-dihydroxyhopane), another triterpene,
has been found in several other species of Hypocrella and
Moelleriella (Isaka et
al. 2003). Interestingly, these triterpenes have been found
in Hypocrella and Moelleriella but not in other
entomopathogenic fungi (Isaka et
al. 2003).
Hypocrella/Aschersonia, Samuelsia, and Moelleriella in biological control of insects
The ability of Hypocrella, Samuelsia, and Moelleriella to
cause epizootics on whitefly and scale-insect populations makes these three
genera potentially useful agents of biological control. The first biocontrol
applications were done with Moelleriella libera (anam.
"A." aleyrodis) to control citrus whitefly in Florida,
U.S.A. (Berger 1921). In some
regions, whiteflies are still effectively controlled by epizootics following
these original applications (Samson &
Rombach 1985). Rolfs and Fawcett
(1908) included two
Moelleriella species (then classified in Aschersonia) among
the so-called "friendly" fungi. Their "red fungus" is
M. libera. Their "yellow fungus" was thought to be
Aschersonia flavocitrina; however Petch
(1921) reported that it was
A. goldiana Sacc. & Ellis. Aschersonia goldiana is now
considered a synonym of M. libera, and modern collections from
Florida have also been shown to be a yellow-spored form of M. libera
(Liu et al.
2006).
Interest in the use of Aschersonia species as biocontrol agents started to decline around the 1920s, following doubts about their efficacy and the increasing popularity of chemical insecticides (Evans & Hywel-Jones 1990). In the 1960s and 1970s, biological control research on Aschersonia s. l. species was revived, especially in Eastern Europe and Asia (Evans & Hywel-Jones 1990). Since then, many studies have shown that aschersonia-like species can been successful biocontrol agents against several species of whiteflies and scale insects (Uchida 1970, Ferron 1978, Ramakers 1983, Ramakers & Samson 1984, Hall 1985, Gerling 1986, Fransen 1987, Gerling 1992, Meekes et al. 1996, Faria & Wraight 2001, Meekes 2001, Sengonca et al. 2006, among others). Meekes (2001) presented an extensive list of Aschersonia s. l. species and susceptible insect hosts (Table 1.1., p. 5, in Meekes 2001). Because Samuelsia is a small genus with few known collections, no information on its biocontrol potential exists.
Hypocrella and Moelleriella meet some of the important criteria for the development of a mycoinsecticide: ease of production in artificial medium, high spore yield, and high virulence against the target insect. In general, Hypocrella and Moelleriella species readily produce conidia in artificial media. Light may enhance conidial production (Hirte et al. 1989, Ibrahim et al. 1993, Lacey et al. 1996, Meekes 2001). However, the fact that most species of these two genera do not sporulate in liquid culture, or, if so, only at its surface, is an impediment to mass production for biocontrol (Ibrahim et al. 1993). In addition, the lack of dispersing agents in greenhouses (e.g. wind and rain) makes repeated applications necessary to maintain high levels of the pathogen in the insect population (Samson & Rombach 1985).
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MATERIALS AND METHODS |
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Cultures were obtained by isolating asci containing ascospores or conidial masses, and placing them on Difco potato dextrose agar (PDA) with a 1 % antibiotic solution (0.2 % Sigma Streptomycin Sulfate + 0.2 % Sigma Neomycin Sulfate). Additional cultures were obtained from ARS Collection of Entomopathogenic Fungal Cultures, New York, U.S.A. (ARSEF); Biotec Culture Collection, Bangkok, Thailand (BCC); and Centraalbureau voor Schimmelculures, Utrecht, The Netherlands (CBS).
Morphological characterisation
For morphological characterisation, the macromorphology of the stroma was
observed: location of the stromata on the plant (i.e. abaxial or
adaxial surface of leaves, stems, leaf veins), stroma size, colour, shape,
hardness, presence/absence of hypothallus, texture of the stroma surface,
distribution of perithecia and conidiomata in the stroma, presence of
perithecia and conidiomata in the same stroma, presence of projecting or
embedded perithecia, colour of ostiole openings, colour of conidiomatal
cavities or openings, and colour of conidial masses. Colour terminology is
from Kornerup and Wansher
(1967). The reaction of the
stromatal tissue in 3 % potassium hydroxide (KOH) was noted. If a change in
colour was observed then it is noted as KOH+. To observe internal
characteristics of the stromata, such as tissue type, diameter of stromal
hyphae, and shape of perithecia, the stromata were rehydrated briefly in
distilled water with a trace of Tween® 80 (J.T. Baker Chemical Co.,
Phillipsburg, New Jersey, U.S.A.). Then, the rehydrated stromata were
supported by Tissue-Tek O.C.T. Compound 4583 (Miles Inc., Elkhart, Indiana,
U.S.A.) and sectioned at a thickness of ca. 15 µm with a freezing
microtome. Characteristics of the asci, ascospores, phialides, paraphyses, and
conidia were observed by rehydrating the stroma in 3 % KOH or water containing
Tween® and removing part of the centrum with a fine glass needle and
placing it on a glass slide. Terminology applying to stromal tissue types
(i.e. textura angularis, intricata, oblita, epidermoidea) is used in
the sense of Korf (1958).
Morphological observations of the colonies and anamorphs in culture were based
on cultures grown on PDA for ca. 3 wk in an incubator at 25 °C
with alternating 12 h fluorescent light and 12 h darkness. Based on Liu &
Hodge (2005), growth rate over
3 wk has been categorised as: fast-growing 30-35 mm diam, moderate growth
20-30 mm diam, and slow-growing <20 mm diam.
Measurements of continuous characters such as length and width were made
using the beta 4.0.2 version of Scion Image software (Scion Corporation,
Frederick, Maryland, U.S.A.). Confidence intervals ( = 0.05), minimum
and maximum values for 10-30 anamorph and teleomorph measurements (except
where indicated) were calculated using Systat 8.0 (SPSS, Inc., Chicago,
Illinois, U.S.A.).
DNA extraction, PCR and sequencing
Cultures of Hypocrella, Samuelsia, and Moelleriella
species used in the phylogenetic analyses
(Table 2) were grown on
potato-dextrose broth in a 6-cm-diam Petri plate for about 1 wk. The mycelial
mat was harvested in a laminar flow hood and then dried using clean, absorbent
paper towels. DNA was extracted with Ultra CleanTM Plant DNA Isolation
Kit (MO BIO Laboratories, Inc., Solana Beach, California, U.S.A.). To extract
DNA from herbarium specimens, the surface of the stroma was first cleaned
briefly with sterilised distilled water, then rehydrated by placing the stroma
in a small Petri plate with sterilised distilled water and letting it stand
for a few minutes until the stroma became softer. Subsequently, a very thin
layer of the surface of the stroma was shaved off using a scalpel and then
discarded. Pieces of the clean inner stroma, including centri, were cut out
and then placed in a 1.5-mL Eppendorf tube for immediate DNA extraction with
Ultra CleanTM Plant DNA Isolation Kit.
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Three partial gene regions were amplified, i.e., large subunit
nuclear ribosomal DNA (LSU), translation elongation factor 1-
(EF1-), and RNA polymerase II subunit one (RPB1). The primers used were
LSU: LRORf (5'-GTACCCGCTGAACTTAAGC-3') and LR5r (5'-ATCCTGAGGGAAACTTC-3')
(Vilgalys & Hester 1990);
EF1-: 983f (5'-GCYCCYGGHCAYCGTGAYTTYAT-3')
(Carbone & Kohn 1999) and
2218r (5'-ATGACACCRACRGCRACRGTYTG-3')
(Rehner 2001); RPB1: cRPB1Af
(5'-CAYCCWGGYTTYATCAAGAA-3') and RPB1Cr (5'-CCNGCDATNTCRTTRTCCATRTA-3')
(Castlebury et al.
2004). Each 50 µL-PCR reaction contained 25 µL of Promega 2X
PCR Master Mix (Promega Corporation, Madison, Wisconsin, U.S.A.), 2.5 µL of
each forward and reverse primers (10 mM), 1 µL DMSO (dimethyl sulfoxide),
ca. 25 ng of genomic DNA, and sterile distilled water. The PCR
reactions were placed in an Eppendorf Mastercycler thermocycler (Eppendorf,
Westbury, New York, U.S.A.) under the following conditions: for LSU (1) 5 min
at 94 °C, (2) 35 cycles of denaturation at 94 °C for 30 s, annealing
at 50 °C for 45 s, and extension at 72 °C for 1 min, (3) and 7 min at
72 °C; for EF1- (1) 10 min at 95 °C, (2) 40 cycles of
denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and
extension at 72 °C for 1 min, (3) and 72 °C for 10 min; and for RPB1
(1) 5 min at 95 °C, (2) 40 cycles of denaturation at 95 °C for 1 min,
annealing at 50 °C for 2 min, and extension at 72 °C for 2 min, (3)
and 72 °C for 10 min. The resulting PCR products were purified with the
QIAquickTM PCR Purification Kit (Qiagen, Inc., Valencia, California,
U.S.A.). Sequencing of forward and reverse strands was performed at the DNA
Sequencing Facility (Center for Agricultural Biotechnology, University of
Maryland, College Park, Maryland, U.S.A.). Sequences were assembled and edited
with Sequencher 4.2 (Gene Codes, Madison, Wisconsin, U.S.A.). Sequences have
been deposited in GenBank (Table
2).
Phylogenetic analyses
The sequences produced were aligned with Clustal x 1.81
(Thompson et al.
1997) and the alignment was refined by hand with GeneDoc 2.6.002
(Nicholas et al.
1997). Maximum Parsimony (MP), Neighbor Joining (NJ), and Bayesian
Inference (BI) analyses were carried out with all sequences. The MP analysis
was done in PAUP v. b10 (Swofford
2002) using a heuristic search, with a starting tree obtained via
1000 random stepwise addition sequences, tree-bisection-reconnection as the
branch-swapping algorithm, and MULTREES off. Neighbour-Joining analyses were
done using the Kimura 2-parameter model. Bootstrap values (BP) from 1000
replicates were calculated for both MP and NJ. MrBayes 3.0 b4
(Huelsenbeck 2000,
Huelsenbeck et al.
2001) was used to reconstruct phylogenetic trees based on the
Bayesian approach (Rannala & Yang
1996, Mau et al.
1999). The Bayesian analysis used a different model of evolution
for each of the three partitions (LSU, TEF, RPB1). The models of DNA
substitution were estimated using Modeltest 3.6
(Posada & Crandall 1998)
and are detailed in Chaverri et al.
(2005a). Four chains and
5,000,000 Markov Chain Montecarlo generations were run and the current tree
was saved to a file every 100 generations. Stability of likelihood scores was
confirmed using the software TRACER version 1.2.1
(Rambaut & Drummond
2007), which traces the parameter against the generation number.
Once stability was reached both in terms of likelihood scores and parameter
estimation, the first 5,000 trees were discarded ("burn in"). The
remaining trees ("post-burn in") were pooled and a 50 %
majority-rule consensus tree was obtained with PAUP*.
Epichloë elymi Schardl & Leuchtm., Balansia
henningsiana (A. Möller) Diehl, and Regiocrella sinensis
Chaverri & K.T. Hodge were used as outgroup species. The close
relationship of the selected outgroup species to Hypocrella,
Moelleriella, and Samuelsia was shown in Chaverri et
al. (2005a).
Topological incongruence was examined using a reciprocal 70 % bootstrap
(BP) or a 95 % posterior probability (PP) threshold
(Mason-Gamer & Kellogg
1996, Reeb et al.
2004) in order to determine whether the sequences from the three
genes should be combined in a single analysis. Bootstrap values were generated
using NJ with 1000 replicates and a maximum likelihood distance. Posterior
probabilities were calculated using Bayesian analysis in MrBayes. A conflict
was assumed to be significant if two different relationships for the same
taxa, one being monophyletic and the other non-monophyletic, both with BP
70 % and PP 95 %, were observed on each LSU, EF1-, and RPB1
majority-rule consensus trees. The three partitions could be combined if no
significant conflicts were detected.
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RESULTS |
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TOPAbstractINTRODUCTIONTHE GENERA...MATERIALS AND METHODSRESULTSDISCUSSIONKEY TO GENERA TREATEDSYNOPTIC KEYSDICHOTOMOUS KEYSGENERA AND SPECIES DESCRIPTIONSEXCLUDED OR DOUBTFUL SPECIESReferences |
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, and 741 from RPB1
(Table 3). RPB1 contributed
more polymorphic sites (46 %), than EF1- (36 %) and LSU (25 %). The
amount of homoplasious characters (i.e. parallel, convergent,
reversed, or superimposed changes) was relatively high, especially in
EF1- and RPB1 data (Table
3). Whelan et al.
(2001) reported that as
homoplasy levels increase the likelihood of finding the correct evolutionary
tree using MP is progressively reduced. This was noticed in the individual LSU
(Fig. 1) and EF1- trees
where BP values for MP analyses were low, especially at the backbone of the
tree, and fewer clades were supported by BP values >75 % (EF1- and
RPB1 trees not shown; BP data indicated in
Fig. 2). The high homoplasy
especially affects the EF1- MP tree where low BP values support the
internal nodes, thus reducing the effective resolution. Therefore, NJ and BI
trees will add confidence to the resulting phylogeny. RPB1 sequence data
produced a well-resolved tree with high BP values at internal and external
nodes. LSU, EF1-, RPB1, and combined data trees, show high BP support
for the species treated in this study. However, only EF1- and RPB1 data
were able to distinguish between the disjunct sister species M.
ochracea (New World) and M. mollii (Old World).
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Hypocrella/Aschersonia, Samuelsia, and Moelleriella
The results of the phylogenetic analyses illustrate three major clades: one
that includes species with non-disarticulating ascospores and fusiform conidia
(i.e. Hypocrella), a second one that includes species with
non-disarticulating ascospores and allantoid small conidia (i.e.
Samuelsia), and a third one that includes species with disarticulating
ascospores and fusiform conidia (i.e. Moelleriella) (Figs
1-2).
The MP and NJ analyses of LSU sequence data supports Moelleriella
with MP BP of <50 % and NJ BP of 74 %; Samuelsia with MP BP of 64
% and NJ BP of 71 %; and Hypocrella with MP BP of 89 % and NJ BP of
96 %. Similarly, MP and NJ analyses of RPB1 also support these three major
clades/genera: Moelleriella is supported by MP BP of 61 % and NJ BP
of 97 %, Samuelsia by MP BP of <50 % and NJ BP of 61 %, and
Hypocrella by MP BP of 93 % and NJ BP of 100 %. The low bootstrap
values at the internal nodes in EF1- MP and NJ trees do not support
these groups. In the combined analyses
(Fig. 2), Moelleriella
is supported by MP BP 63 %, NJ BP 98 %, and Bayesian Inference (BI) posterior
probability (PP) of 100 %; Samuelsia by MP BP 67 %, NJ BP 86 %, and
PP of 100 %; and Hypocrella by MP BP 100 %, NJ BP 100 %, and BI PP of
100 %.
RPB1 and the combined phylogenetic trees show high BP and PP support for
the three subclades of Hypocrella s. l. that were reported by
Chaverri et al.
(2005b): Pulvinate, Globose,
and Effuse (Fig. 2).
Moelleriella can be divided into the Effuse and Globose clades. The
Effuse clade includes M. basicystis, M. disjuncta, M. evansii, M.
libera, M. madidiensis, M. mollii, M. ochracea, M. rhombispora, M. phyllogena,
M. raciborskii, M. umbospora, and M. zhongdongii. The Globose
clade includes M. africana, M. boliviensis, M. epiphylla, M. insperata, M.
macrostroma, M. schizostachyi, M. sloaneae, and M. turbinata.
The Pulvinate clade (i.e. Hypocrella s. str. and Samuelsia)
includes two groups: Pulvinate A and Pulvinate B. Pulvinate A is
Hypocrella s. str. and contains H. citrina, H. hirsuta, H.
disciformis, H. viridans, and H. discoidea; and Pulvinate B is
Samuelsia and includes S. chalalensis, S. geonomis, S.
rufobrunnea, and S. sheikhii. EF1- data do not support
those three groups and LSU data show relatively weak support only for the
Pulvinate clade. The Hypocrella s. str. clade, and the nodes nested
within, have better resolution, BP and PP support, than the Effuse and Globose
clades and nodes within Moelleriella. Some polytomies are observed
within the Effuse clade.
Moelleriella
In the combined analyses (Fig.
2), the close relationship between the disjunct sister taxa M.
ochracea (NW) vs. M. mollii (OW) and M. libera (NW) vs.
M. raciborskii (OW) is clear. The M. libera clade is
supported by MP BP of 90 %, NJ BP of 100 %, and PP of 61 %. The M.
raciborskii clade is supported by MP BP of 99 %, NJ BP of 100 %, and PP
of 100 %. The node that supports these two taxa has a MP and NJ BP, and PP of
100 %. Moelleriella evansii, which is morphologically similar to
M. libera, is basal to a robust clade that includes M.
libera and M. raciborskii (Figs
1-2).
Likewise, the M. ochracea clade is supported by high MP BP, NJ BP,
and PP values (91, 99, and 100 %, respectively). A M. mollii clade is
supported by 100 % BP and PP values. The node that includes M. mollii
and M. ochracea is supported by 100 % BP and PP values.
Moelleriella mollii and M. ochracea are within a clade that
includes two other morphologically similar taxa: M. zhongdongii and
M. madidiensis. This clade is supported by BP of 78 %, NJ BP of 100
%, and PP of 100 %.
Moelleriella phyllogena (type species of Moelleriella), M. umbospora, M. basicystis, and M. disjuncta are morphologically similar species that belong to a clade supported by high BP and PP values (Fig. 2). The structure of this clade correlates with geographic origin: M. basicystis in Panama and Costa Rica (probably southern Central America); M. phyllogena in Panama, Brazil, Ecuador, Bolivia, and Peru (probably Panama and Amazon basin); M. umbospora in Mexico, Honduras, Guatemala (probably northern Central America); and M. disjuncta in Panama and Guyana. Recognition of each of these species is supported by high BP and PP values. Moelleriella disjuncta is basal within this clade. Although M. rhombispora shares some morphological similarities to the M. phyllogena species complex, DNA sequence data do not support their close relationship.
Combined DNA sequence analyses show significant support for most nodes (internal and external) within the Globose clade (Fig. 2). Moelleriella epiphylla and M. turbinata are morphologically similar species and their close relationship is supported by MP BP of 78 %, NJ BP of 95 %, and PP of 100 %. The M. epiphylla and M. turbinata clades are closely related to a group that contains mostly species with relatively large, globose and hard stromata (except M. insperata): M. boliviensis, M. africana, M. schizostachyi, and M. macrostroma. Species with large stromata are closely related (MP BP 78 %, NJ BP 96 %, and PP 100 %). Moelleriella sloaneae is basal in the Globose clade and is morphologically different from other species in the group. The inclusion of M. sloaneae in the Globose group is weakly supported in the combined phylogenetic analyses: MP BP <50 %, NJ BP 75 %, and PP 100 %. Only RPB1 data shows some support for this relationship (MP BP of 53 %, NJ BP of 88 %).
Hypocrella/Aschersonia s. str.
This clade includes species with non-disarticulating ascospores and
fusiform conidia, thus Hypocrella s. str. All nodes in this group are
strongly supported by BP and PP values
(Fig. 2). Hypocrella
citrina, H. disciformis, H. hirsuta, and H. viridans are
morphologically similar species, are widespread in the Neotropics, and belong
to a robustly supported clade (MP BP 78 %, MP NJ 100 %, PP 100 %). The
Hypocrella discoidea (type species of Hypocrella) species
complex is common throughout the Old World, is closely related and
morphologically similar to Hypocrella citrina, H. disciformis, H.
hirsuta, and H. viridans.
Samuelsia
This clade includes species with non-disarticulating ascospores and small
allantoid conidia. The species contained in this clade are rare and probably
endemic, i.e. S. rufobrunnea (type species of Samuelsia)
(Bolivia, Peru), S. chalalensis (Bolivia), S. geonomis
(Bolivia), and S. sheikhii (Honduras). The three Bolivian species are
morphologically similar and belong in the same monophyletic group (BP and PP
of 100 %).
Morphological analyses and geographical distribution
Moelleriella, Samuelsia, and Hypocrella/Aschersonia
The distributions of the species presented here are generally poorly known.
Based on the specimens collected for this study, Moelleriella is most
diverse and common in the Neotropics (22 known species), followed by
Hypocrella (5 known species), and Samuelsia (5 known
species). Moelleriella and Hypocrella species are also more
abundant that Samuelsia. Field collections and additional specimens
from collaborators and herbaria yielded hundreds of specimens of
Moelleriella and Hypocrella from many different countries.
On the other hand, Samuelsia species are only known from one
collection each. Samuelsia rufobrunnea, S. chalalensis, and S.
geonomis are known from Bolivia and Peru; S. sheikhii from
Honduras; and S. intermedia from Chile.
Examination of multiple morphological characters shows that segmentation of ascospores and conidial size and shape are the main characters that distinguish Moelleriella, Samuelsia, and Hypocrella. All known species of Moelleriella have filiform multiseptate ascospores that disarticulate at maturity inside the ascus and fusiform conidia < 15 µm long. Hypocrella species have filiform to long-fusiform multiseptate ascospores that do not disarticulate and fusiform conidia > 15 µm long. Samuelsia species have long-fusiform, non-disarticulating ascospores and small allantoid conidia < 10 µm long. Another character common to most species of Hypocrella and Samuelsia is a reddish colour reaction of the stroma when 3 % KOH is applied. However, H. citrina does not change colour. All studied species of Moelleriella lack a reaction to 3 % KOH. Moelleriella gaertneriana and M. cornuta release brownish pigments when 3 % KOH is added, but the tissue of the stromata does not change colour. Stromal anatomy is somewhat conserved in Hypocrella. The Hypocrella species examined have pulvinate stromata, with or without hypothalli. On the other hand, stromata in Moelleriella are highly variable: from flat or effuse with loose hyphal tissue (e.g. M. libera), to somewhat pulvinate (e.g. M. madidiensis) or knob-shaped (e.g. M. phyllogena, M. basicystis, M. umbospora, M. disjuncta), to large, globose and hard stromatal tissue (e.g. M. macrostroma, M. gaertneriana).
Characters of the anamorph are somewhat useful in distinguishing between Hypocrella, Samuelsia, and Moelleriella. The three genera have pycnidial-acervular conidiomata, slender flask-shaped to cylindrical phialides arranged in a compact hymenium, with or without paraphyses, brightly coloured and slimy conidial masses, and fusoid, unicellular conidia. Only conidial shape and size are useful characters.
Moelleriella morphology
The stromatal size of Moelleriella species treated in this
study ranges from 0.5-30 mm diam. Moelleriella macrostroma, M.
gaertneriana, and M. cornuta have large stromata that range from
2-30 mm diam. The smallest stromata are those of M. umbospora, M.
basicystis, M. turbinata, M. castanea, and M. colliculosa, which
range between 0.5 and ca. 2 mm in diam. The size of the stroma is
generally correlated with the number of perithecia or pycnidia per stroma. The
colours of the stromata of the species studied are in shades of white,
yellow, orange, brown, or black. White and orange are the most common colours.
A few species, i.e. M. cornuta, M. turbinata, M. palmae, M. guaranitica,
M. globosa, and M. castanea, have brown to almost black
stromata; these species are phylogenetically related in the Globose clade
(Figs
1-2).
Most of the examined species in the Effuse clade have stromata in shades of
white or orange. The shapes of the stromata are highly variable. They
can range from effuse or flat, to somewhat pulvinate, globose, cerebriform,
tubular, conical, or knob-like. Moelleriella libera, M. ochracea, M.
sloaneae, and M. evansii have effuse or thin, pulvinate
stromata. Moelleriella zhongdongii, M. madidiensis, M. rhombispora, M.
guaranitica, M. castanea, and M. epiphylla, have pulvinate to
convex stromata. The surface of the stroma can be shiny or dull/matt,
opaque, glabrous or scurfy, and tomentose or roughened. Most species of
Moelleriella have a stromatal surface that is opaque and pruinose;
however, there are many exceptions. Moelleriella libera, M. evansii, M.
zhongdongii, M. madidiensis, M. ochracea, M. rhombispora, M. sloaneae,
and some forms of M. basicystis, M. disjuncta, M. phyllogena, and
M. umbospora, have tomentose stroma surfaces. Teleomorph stromata of
M. basicystis, M. disjuncta, M. phyllogena, and M.
umbospora, and stromata of M. macrostroma, M. gaertneriana, M.
boliviensis, M. turbinata, M. epiphylla, M. cornuta, M. palmae, M. globosa, M.
guaranitica, M. castanea, and M. colliculosa have almost
glabrous surfaces. Stromata of M. macrostroma, M. gaertneriana, and
M. cornuta, have somewhat shiny stromata. hypothalli are
present in some species, except M. epiphylla, M. turbinata, M.
boliviensis, M. macrostroma, and M. gaertneriana, M. castanea, M. colliculosa,
M. globosa, and M. guaranitica. There is no reaction to 3 %
KOH on the tissue of the stromata; only M. gaertneriana and
M. cornuta release brownish pigments when KOH is added. The tissue
structure of the stroma surface is highly conserved among the examined
species. All species studied have the tissue type referred to as textura
intricata or epidermoidea. Some differences are found in the
thickness of the cell walls. Darkly pigmented hard stromata are composed of
hyphae with thick cell walls (ca. 2-6 µm). Lightly pigmented
stromata with loose hyphal tissues have thin-walled hyphae (ca. 1
µm). The inner tissue below the outermost layer of the stroma is
generally of textura oblita, intricata, epidermoidea or a condition
intergrading between these tissue types.
Perithecia in some species of Moelleriella can be formed in well-separated or gregarious tubercles, or they can be completely embedded in the stroma. Moelleriella libera, M. ochracea, M. rhombispora, M. zhongdongii, M. evansii, and M. sloaneae form perithecial tubercles. When the stroma contains perithecia and pycnidia, the perithecia are either towards the edges of the stroma or scattered, with pycnidia either clustered in the centre or interspersed among the perithecia, respectively. The ostioles can range in colour from yellow to orange yellow, to reddish brown or brownish yellow. In longitudinal section, perithecia are generally elongated to subglobose, sometimes globose, with walls composed of 3-4 layers of highly compacted, thin-walled cells.
The asci are cylindrical and sometimes swollen in the middle when ascospores have disarticulated and accumulated towards the middle. Generally the ascus cap is thickened (ca. 2-5 µm) and capitate. Moelleriella boliviensis, M. epiphylla, M. globosa, M. guaranitica, M. colliculosa, and M. castanea have thin ascus tips (< ca. 1.5 µm). Because the multiseptate filiform ascospores disarticulate at the septum inside the ascus upon mounting, it is difficult to know exactly the length of intact ascospores. The part-ascospores are always hyaline and smooth. They can vary in shape, from cylindrical, to fusoid, ventricose, or almost ovoid. Moelleriella basicystis, M. phyllogena, M. umbospora, and M. disjuncta have a swollen protuberance on one side of the part-spore. In M. rhombispora, the part-spores have similar protuberances in the middle (i.e. ventricose shape). For the most part, the species studied have part-spo
