The gut of Guatemalan passalid beetles: a habitat colonized by cellobiose- and xylose-fermenting yeasts

The gut of insects is a productive environment for discovering undescribed species of yeasts, and the gut of wood-feeding insects of several families is especially rich in yeasts that carry out the fermentation of cellobiose and xylose. Passalid beetles (Passalidae, Coleoptera) live in dead wood that they ingest as their primary food source. We report the isolation, molecular identification and physiological characterization of 771 yeast cultures isolated from the gut of 16 species of passalids collected in nine localities in Guatemala. Ascomycete yeasts were present in the gut of every passalid studied, and the xylose-fermenting (X-F) yeasts Scheffersomyces shehatae and Scheffersomyces stipitis were the most abundant taxa isolated. The gut of the beetles also contained undescribed cellobiose-fermenting and X-F species in the Lodderomyces, Scheffersomyces and Spathaspora, and undescribed species in Sugiyamaella clades as well as rare yeast species in the Phaffomyces and Spencermartinsiella clades. Basidiomycete yeasts in the genera Cryptococcus and Trichosporon were also common. The yeast species richness was influenced by the host species and the substrate, and gut-inhabiting yeasts have the ability to survive the differing physiological conditions of several gut compartments.     

Study on the effects of near-future ocean acidification on marine yeasts: a microcosm approach

Marine yeasts play an important role in biodegradation and nutrient cycling and are often associated with marine flora and fauna. They show maximum growth at pH levels lower than present-day seawater pH. Thus, contrary to many other marine organisms, they may actually profit from ocean acidification. Hence, we conducted a microcosm study, incubating natural seawater from the North Sea at present-day pH (8.10) and two near-future pH levels (7.81 and 7.67). Yeasts were isolated from the initial seawater sample and after 2 and 4 weeks of incubation. Isolates were classified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and representative isolates were identified by partial sequencing of the large subunit rRNA gene. From the initial seawater sample, we predominantly isolated a yeast-like filamentous fungus related to Aureobasidium pullulans, Cryptococcus sp., Candida sake, and various cold-adapted yeasts. After incubation, we found more different yeast species at near-future pH levels than at present-day pH. Yeasts reacting to low pH were related to Leucosporidium scottii, Rhodotorula mucilaginosa, Cryptococcus sp., and Debaryomyces hansenii. Our results suggest that these yeasts will benefit from seawater pH reductions and give a first indication that the importance of yeasts will increase in a more acidic ocean.     

Cryptococcus nanyangensis sp. nov., A New Basidiomycetous Yeast Isolated from the Gut of Wood-Boring Larvae

Two strains of a novel basidiomycetous yeast species were isolated from the gut of wood-boring larvae collected in the Baotianman Nature Reserve, the central China. Sequence analysis of the D1/D2 domains of the large subunit (LSU) rRNA gene and internal transcribed spacer (ITS) regions showed that these yeasts belong to the Bulleromyces clade and formed a cluster together with eleven undescribed Cryptococcus species. The novel species differed from its closest known species, Cryptococcus rajasthanensis, by 3.3 % divergence (15 substitutions and 6 gaps over 630 bases) in the D1/D2 domains, and by 13.4 % divergence (41 substitutions and 27 gaps over 508 bases) in the ITS regions. Physiologically, the fermentation of glucose, galactose, sucrose, trehalose, and raffinose in Durham tubes was observed for the strains of this new yeast. Based on the phenotypical and molecular characteristics presented, the two strains are proposed as a new species, Cryptococcus nanyangensis sp. nov., with the type strain KCY-1^T (=CICC 1976^T = CBS 12474^T).     

Yeast communities of the cactusPilosocereus arrabidae and associated insects in the Sandy Coastal Plains of Southeastern Brazil

The yeast communities from necrotic tissues, decaying flowers and fruits, and from larval feeding sites of the mothSigelgaita sp. in the cactusPilosocereus arrabidae were surveyed in three restinga ecosystems in Southeastern Brazil. Insects associated with these substrates were sampled to verify the vectoring of yeasts. The cactusPilosocereus arrabidae was shown to have four different yeast communities associated with it. Necrotic stems had a diverse yeast community with the prevalent speciesPichia barkeri, Candida sonorensis, Pichia cactophila, Geotrichum sp.,Myxozyma mucilagina andSporopachydermia sp. A, representing about 80% of the total isolates.Pichia sp. A and aCandida domercqii-like species represented more than 90% of the yeast isolates from decaying flowers. Fruits had a heterogeneous yeast community with typical fruit yeasts of the genusKloeckera, basidiomicetous anamorphs of the genusCryptococcus, the black yeastAureobasidium pullulans, Pichia sp. A, aCandida domercqii-like species, and some cactophilic yeasts, especiallyClavispora opuntiae. The feeding site ofSigelgaita sp. larvae hadClavispora opuntiae as the prevalent species. Insect vectors are suggested as one the most important factors influencing the composition of these yeast communities.     

Yeast diversity in the acidic Rio Agrio-Lake Caviahue volcanic environment (Patagonia, Argentina)

The Rio Agrio and Lake Caviahue system (RAC), in Northwestern Patagonia, is a natural acidic environment. The aims of this study were to characterize the yeast community and to provide the first ecological assessment of yeast diversity of this extreme aquatic environment. Yeast occurrence and diversity were studied at seven sites where the water pH varied between 1.8 and 6.7. Yeast CFU counts in the river ranged from 30 to 1200 CFU L⁻¹, but in the Lake the values were lower (30–60 CFU L⁻¹). A total of 25 different yeast species were found, 11 of which belonged to undescribed taxa. Among these was an unusual strongly acidophilic Cryptococcus species. The RAC yeast community resembles that of acidic aquatic environments resulting from anthropic activities such as the São Domingos mines in Portugal and the Rio Tinto in Spain, respectively. The isolated yeast species were organized into different grades of adaptation to the RAC aquatic system. Based on the proposed grades, Rhodotorula mucilaginosa , Rhodosporidium toruloides and two novel Cryptococcus species were the most adapted species. These Cryptococcus species are apparently specialists of acidic aquatic environments, and might bear physiological features that possibly account for their ability to thrive in such extreme environments.     

Species Richness of Yeast Communities in Floral Nectar of Southern Spanish Plants

Floral nectar of insect-pollinated plants often contains dense yeast populations, yet little quantitative information exists on patterns and magnitude of species richness of nectar-dwelling yeasts in natural plant communities. This study evaluates yeast species richness at both the plant community and plant species levels in a montane forest area in southern Spain, and also explores possible correlations between the incidence of different yeast species in nectar and their reported tolerance to high sugar concentrations, and between yeast diversity and pollinator composition. Yeast species occurring in a total of 128 field-collected nectar samples from 24 plant species were identified by sequencing the D1/D2 domain of the large subunit rDNA, and rarefaction-based analyses were used to estimate yeast species richness at the plant community and plant species levels, using nectar drops as elemental sampling units. Individual nectar samples were generally characterized by very low species richness (1.2 yeast species/sample, on average), with the ascomycetous Metschnikowia reukaufii and Metschnikowia gruessii accounting altogether for 84.7% of the 216 isolates identified. Other yeasts recorded included species in the genera Aureobasidium, Rhodotorula, Cryptococcus, Sporobolomyces, and Lecythophora. The shapes and slopes of observed richness accumulation curves were quite similar for the nectar drop and plant species approaches, but the two approaches yielded different expected richness estimates. Expected richness was higher for plant species-based than for nectar drop-based analyses, showing that the coverage of nectar yeast species occurring in the region would be improved by sampling additional host plant species. A significant correlation was found between incidence of yeast species in nectar and their reported ability to grow in a medium containing 50% glucose. Neither diversity nor incidence of yeasts was correlated with pollinator composition across plant species.     

Taxonomic and phenotypic characterization of yeasts isolated from worldwide cold rock-associated habitats

Yeast strains isolated from rock samples collected from worldwide cold regions were identified by sequence analysis of the D1/D2 domains of the 26S rDNA gene and the ITS region followed by molecular phylogeny. Over 77 % of yeasts isolates were Basidiomycota. Cryptococcus (orders Filobasidiales and Tremellales) and Rhodotorula (order Cystobasidiales) were the most frequent genera. About 40 % of yeast isolates belonged to undescribed species.     

The ecology of the yeast flora in necroticOpuntia cacti and of associatedDrosophila in Australia

A survey was made of the yeast communities isolated from necrotic tissue of 4 species of prickly-pear cacti (Opuntia stricta, O. tomentosa, O. monacantha, andO. streptacantha) which have colonized in Australia. Yeast communities were sampled from a number of localities and at different times. Cactus specific yeasts accounted for 80% of the total isolates, and the 3 most common species contributed 63% of the total. Comparisons of the species compositions of the yeast communities indicated that the differences among communities were greater betweenOpuntia species than between different localities within a single cactus species, and also that differences between years were greater than average differences between localities within years. Multivariate statistical tests of association between yeast community and physical features of rots indicated that temperature, pH, and age of rot all exerted some influence on the structure of the yeast community. Similar analyses involvingDrosophila species inhabiting these cactus rots suggested the existence of complex associations betweenDrosophila community, yeast community, and physical and chemical attributes of the cactus necroses.     

Microeukaryotic diversity in the extreme environments of the Iberian Pyrite Belt: a comparison between universal and fungi-specific primer sets, temperature gradient gel electrophoresis and cloning

The Iberian Pyrite Belt extends from Portugal to Spain and is one of the most important pyrite regions in the world. Its aquatic reservoirs display extreme conditions characterized by low pH and high concentrations of heavy metals. In this study, the diversity of microeukaryotes was analysed at the abandoned mines of S?o Domingos (Portugal) and at Rio Tinto (Spain). DNA was extracted from water samples and a set of eukaryotic universal primers directed to the small subunit rRNA genes (rDNA) was used. The amplicons were analysed by molecular cloning and temperature gradient gel electrophoresis (TGGE). In addition, a fungi-specific primer set was also used in TGGE experiments. The fungi-specific primers contributed to a substantial increase in the number of fungal taxa found due, probably, to the relative low density of fungal structures. Several microorganisms, belonging (or closely related) to the ascomycetous yeast Pichia acaciae, the basidiomycetous yeasts Cryptococcus humicola and Cystofilobasidium bisporidii, the green algae Chlamydomonas noctigama and Chlorella protothecoides var. acidicola and some uncultured microeukaryotes were present at both localities, which suggests that specific microorganisms are adapted to the peculiar conditions of the Iberian Pyrite Belt extreme environments. However, in spite of the similarities, a higher algal richness was observed at S. Domingos, whereas for R. Tinto the richness of fungi was more prominent.     

Taxonomic assessment and enzymes production by yeasts isolated from marine and terrestrial Antarctic samples

The aim of the present study was to investigate the taxonomic identity of yeasts isolated from the Antarctic continent and to evaluate their ability to produce enzymes (lipase, protease and xylanase) at low and moderate temperatures. A total of 97 yeast strains were recovered from marine and terrestrial samples collected in the Antarctica. The highest amount of yeast strains was obtained from marine sediments, followed by lichens, ornithogenic soils, sea stars, Salpa sp., algae, sea urchin, sea squirt, stone with lichens, Nacella concinna, sea sponge, sea isopod and sea snail. Data from polyphasic taxonomy revealed the presence of 21 yeast species, distributed in the phylum Ascomycota (n = 8) and Basidiomycota (n = 13). Representatives of encapsulated yeasts, belonging to genera Rhodotorula and Cryptococcus were recovered from 7 different Antarctic samples. Moreover, Candida glaebosa, Cryptococcus victoriae, Meyerozyma (Pichia) guilliermondii, Rhodotorula mucilaginosa and R. laryngis were the most abundant yeast species recovered. This is the first report of the occurrence of some species of yeasts recovered from Antarctic marine invertebrates. Additionally, results from enzymes production at low/moderate temperatures revealed that the Antarctic environment contains metabolically diverse cultivable yeasts, which could be considered as a target for biotechnological applications. Among the evaluated yeasts in the present study 46.39, 37.11 and 14.43 % were able to produce lipase (at 15 °C), xylanase (at 15 °C) and protease (at 25 °C), respectively. The majority of lipolytic, proteolytic and xylanolytic strains were distributed in the phylum Basidiomycota and were mainly recovered from sea stars, lichens, sea urchin and marine sediments.     

云南高原湖泊杞麓湖冬季可培养酵母菌多样性分析

【目的】探究云南杞麓湖酵母菌群落结构及其与环境因子的相互关系。【方法】采用原位培养方法对杞麓湖14个水样进行酵母菌分离,应用26S r DNA D1/D2区域序列分析,并结合形态及生理生化指标将对分离获得的酵母菌进行鉴定,运用软件bio-dap和Canoco分析酵母菌类群的丰富度及其与环境因子间的相互关系。【结果】从杞麓湖中分离得到321株酵母菌,鉴定为14个属27个种和1个潜在的新类群。Rhodosporidium kratochvilovae和出芽短梗霉(Aureobasidium pullulans)是优势种,分别为总菌株数的29.6%和16.8%。水体总磷含量是影响产色素红冬孢酵母属(Rhodosporidium)分布的重要环境因子,而p H为隐球酵母属(Cryptococcus)分布的一个重要选择条件。【结论】杞麓湖酵母菌具有较为丰富的群落多样性。     

Cryptococcus lacticolor sp. nov. and Rhodotorula oligophaga sp. nov., novel yeasts isolated from the nasal smear microbiota of Queensland koalas kept in Japanese zoological parks

A total of 515 yeast strains were isolated from the nasal smears of Queensland koalas and their breeding environments in Japanese zoological parks between 2005 and 2012. The most frequent species in the basidiomycetous yeast biota isolated from koala nasal passages was Cryptococcus neoformans, followed by Rhodotorula minuta. R. minuta was the most frequent species in the breeding environments, while C. neoformans was rare. Seven strains representing two novel yeast species were identified. Analyses of the 26S rDNA (LSU) D1/D2 domain and nuclear ribosomal DNA internal transcribed spacer region sequences indicated that these strains represent new species with close phylogenetic relationships to Cryptococcus and Rhodotorula. A sexual state was not found for either of these two novel yeasts. Key phenotypic characters confirmed that these strains could be placed in Cryptococcus and Rhodotorula. The names Cryptococcus lacticolor sp. nov. (type strain TIMM 10013^T = JCM 15449^T = CBS 10915^T = DSM 21093^T, DDBJ/EMBL/Genbank Accession No.; AB375774 (ITS) and AB375775 (26S rDNA D1/D2 region), MycoBank ID; MB 802688, Fungal Barcoding Database ID; 3174), and Rhodotorula oligophaga sp. nov. (type strain TIMM 10017^T = JCM 18398^T = CBS 12623^T = DSM 25814^T, DDBJ/EMBL/Genbank Accession No.; AB702967 (ITS) and AB702967 (26S rDNA D1/D2 region), MycoBank ID; MB 802689, Fungal Barcoding Database ID; 3175) are proposed for these new species.     

Identification and characterization of yeasts isolated from sedimentary rocks of Union Glacier at the Antarctica

The study of the yeasts that inhabit cold environments, such as Antarctica, is an active field of investigation oriented toward understanding their ecological roles in these ecosystems. In a great part, the interest in cold-adapted yeasts is due to several industrial and biotechnological applications that have been described for them. The aim of this work was to isolate and identify yeasts from sedimentary rock samples collected at the Union Glacier, Antarctica. Furthermore, the yeasts were physiologically characterized, including the production of metabolites of biotechnological interest. The yeasts isolated that were identified at the molecular level belonged to genera Collophora (1 isolate), Cryptococcus (2 isolates), Sporidiobolus (4 isolates), Sporobolomyces (1 isolate) and Torrubiella (2 isolates). The majority of yeasts were basidiomycetous and psychrotolerant. By cross-test assays for anti-yeast activity, it was determined that Collophora sp., Sporidiobolus salmonicolor, and Sporobolomyces roseus secreted a protein factor that kills Sporidiobolus metaroseus. The colored yeasts Sp. salmonicolor, Sp. metaroseus and Collophora sp. produced several carotenoid pigments that were identified as 2,3 dihydroxy-γ-carotene, -carotene, 4-ketotorulene, torulene β-cryptoxanthin and spirilloxanthin. Concerning analysis of mycosporines, these metabolites were only found in the yeasts Torrubiella sp. and Cryptococcus sp. T11-10-1. Furthermore, the yeasts were evaluated for the production of extracellular hydrolytic activities. Of the twelve activities analyzed, alkaline phosphatase, invertase, gelatinase, cellulase, amylase, and protease enzyme activities were detected. The yeasts Cryptococcus sp. T11-10-1 and Sporidiobolus metaroseus showed the highest number of different enzyme activities.     

Characterisation of the diversity and physiology of cellobiose-fermenting yeasts isolated from rotting wood in Brazilian ecosystems

We investigated the yeast species associated with rotting wood samples obtained from Brazilian ecosystems, with a special focus on cellobiose-fermenting species. About 647 yeast strains were isolated from rotting wood samples collected from the areas of Atlantic rainforest, Cerrado, and Amazonian forest. Eighty-six known species and 47 novel species of yeasts were isolated. Candida boidinii, Cyberlindnera subsufficiens, Meyerozyma guilliermondii, Schwanniomyces polymorphus, Candida natalensis, and Debaryomyces hansenii were the most frequently isolated species. Among the cellobiose-fermenting yeasts, 14 known and three novel yeast species were identified. Scheffersomyces queiroziae, Sc. amazonensis, Yamadazyma sp.1, Hanseniaspora opuntiae, C. jaroonii, and Candida tammaniensis were the main ethanol-producing yeasts. These species also produced an intracellular β-glucosidase responsible for cellobiose hydrolysis. In fermentation assays using a culture medium containing 50 g L^?1 cellobiose, ethanol production was observed in all cases; Sc. queiroziae and Sc. amazonensis showed the highest yield, efficiency, and productivity. Candida jaroonii and Yamadazyma sp.1 strains also showed high efficiency in cellobiose fermentation, while C. tammaniensis and H. opuntiae strains produced low amounts of ethanol. This study shows the potential of rotting wood samples from Brazilian ecosystems as a source of yeasts, including new species as well as those with promising biotechnological properties.     

Application of temperature gradient gel electrophoresis to the study of yeast diversity in the estuary of the Tagus river,Portugal

Temperature gradient gel electrophoresis (TGGE) was employed for the assessment of yeast diversity in the estuary of the Tagus river (Portugal). The molecular detection of yeasts was carried out directly from water samples and, in parallel, a cultivation approach by means of an enrichment step was employed. A nested PCR was employed to obtain a fungal amplicon containing the D2 domain of the 26S rRNA gene. For identification the TGGE bands were extracted, re-amplified, and sequenced. Fourteen fungal taxa were detected and all except one were yeasts. Most yeast sequences corresponded to members of the Ascomycota and only three belonged to the Basidiomycota. Five yeasts (four ascomycetes and one basidiomycete) could not be identified to the species level due to the uniqueness of their sequences. The number of species detected after enrichment was higher than the number of taxa found using the direct detection method. This suggests that some yeast populations are present in densities that are below the detection threshold of the method. With respect to the analysis of the yeast community structure, our results indicate that the dominant populations belong to Debaryomyces hansenii, Rhodotorula mucilaginosa, Cryptococcus longus, and to an uncultured basidiomycetous yeast phylogenetically close to Cr. longus. The combined analysis of direct detection and cultivation approaches indicates a similar community structure at the two sampled sites since nine species were present at both localities.     

Yeast Diversity Associated with Invasive Dendroctonus valens Killing Pinus tabuliformis in China Using Culturing and Molecular Methods

Bark beetle-associated yeasts are much less studied than filamentous fungi, yet they are also considered to play important roles in beetle nutrition, detoxification, and chemical communication. The red turpentine beetle, Dendroctonus valens, an invasive bark beetle introduced from North America, became one of the most destructive pests in China, having killed more than 10 million Pinus tabuliformis as well as other pine species. No investigation of yeasts associated with this bark beetle in its invaded ranges has been conducted so far. The aim of this study was to assess the diversity of yeast communities in different microhabitats and during different developmental stages of Den. valens in China using culturing and denaturing gradient gel electrophoresis (DGGE) approaches and to compare the yeast flora between China and the USA. The yeast identity was confirmed by sequencing the D1/D2 domain of LSU ribosomal DNA (rDNA). In total, 21 species (13 ascomycetes and eight basidiomycetes) were detected by culturing method, and 12 species (11 ascomycetes and one basidiomycetes) were detected by molecular methods from China. The most frequent five species in China were Candida piceae (Ogataea clade), Cyberlindnera americana, Candida oregonensis (Metschnikowia clade), Candida nitratophila (Ogataea clade) and an undescribed Saccharomycopsis sp., detected by both methods. Seven species were exclusively detected by DGGE. Ca. oregonensis (Metschnikowia clade) was the most frequently detected species by DGGE method. Eight species (all were ascomycetes) from the USA were isolated; seven of those were also found in China. We found significant differences in yeast total abundance as well as community composition between different developmental stages and significant differences between the surface and the gut. The frass yeast community was more similar to that of Den. valens surface or larvae than to the community of the gut or adults. Possible functions of the yeast associates are discussed.     

Metschnikowia noctiluminum sp. nov., Metschnikowia corniflorae sp. nov., and Candida chrysomelidarum sp. nov., isolated from green lacewings and beetles

Fourteen yeast isolates belonging to the Metschnikowia clade were isolated from the digestive tracts of lacewings (Neuroptera: Chrysopidae), soldier beetles and leaf beetles (Coleoptera: Cantharidae and Chrysomelidae), and a caddisfly (Trichoptera: Hydropsychidae). The insect hosts were associated with sugary substances of plants, a typical habitat for yeasts in this clade. Based on DNA sequence comparisons and phenetic characters, the yeasts were identified as Candida picachoensis, Candida pimensis, and four undescribed taxa. Among the undescribed taxa, three yeasts were distinguished from one another and from other described taxa by nucleotide differences in the ribosomal DNA repeat, which were sufficient to consider them as new species. Two of the novel yeast species are described as Metschnikowia noctiluminum (NRRL Y-27753^T) and M. corniflorae spp. nov. (NRRL Y-27750^T) based in part on production of needle-shaped ascospores, which are found in most Metschnikowia species. Sexual reproduction was not observed in the third new yeast, Candida chrysomelidarum sp. nov. (NRRL Y-27749^T). A fourth isolate, NRRL Y-27752, was not significantly distinct from Metschnikowia viticola and Candida kofuensis to be described as a new species. Phylogenetic analysis of the D1/D2 loop sequences placed M. noctiluminum within the M. viticola clade, while C. chrysomelidarum was a sister taxon of Candida rancensis. Metschnikowia corniflorae was phylogenetically distinct from other new species and fell outside of the large-spored Metschnikowia group.     

Occurrence and Diversity of Yeasts in the Mangrove Ecosystems in Fujian, Guangdong and Hainan Provinces of China

Mangrove wetland is a unique ecosystem and has rich bioresources. In this article, the roots, stems, branches, leaves, barks, fruits, and flowers from 12 species of the mangrove plants and six species of the accompanying mangrove plants, seawater and sediments in mangrove ecosystems in China were used as sources for isolation of yeasts. A total of 269 yeasts strains were obtained from the samples. The results of routine identification and phylogenetic analysis showed that they belonged to 22 genera and 45 species. Of all the 269 strains, Candida spp. was predominant with the proportion of 44.61%, followed by Kluyveromyces spp. (8.55%), Pichia spp. (7.44%), Kodamaea ohmeri (5.58%), Issatchenkia spp. (4.83%) and Debaryomyces hansenii (4.46%). We also found that strains N02-2.3 and ST3-1Y3 belonged to the undescribed species of Pichia sp. and Trichosporon sp. respectively while strain HN-12 was not related to any known yeast strains. This means that different yeast strains of Candida spp. especially C. tropicalis were widely distributed in the mangrove ecosystems and may have an important role in the mangrove ecosystems. The results also showed that some of them may have potential applications.     

Analysis of the community structure of yeasts associated with the decaying stems of cactus. II.Opuntia species

A survey was made of yeast species associated with the decaying pads of 3 prickly pear cacti (Opuntia phaeacantha, O. ficus-indica, andO. lindheimeri) in Arizona and Texas. Yeast communities from 12 localities were compared among localities, amongOpuntia species, and with previous data on yeast communities associated with columnar cacti. The results indicate thatOpuntia necroses contain relatively more yeast species with broader physiological abilities in their communities than columnar necroses. It is argued that differences in chemistry of the opuntias and columnar forms in concert with the insect vectors specific for these cacti account for the differences in yeast community structure. It is further hypothesized that the differences in yeast community structure have been important in the evolution and maintenance of species diversity forDrosophila species which live in the decaying stems or cladodes of various cacti. Most of the yeast community evolution in the cacti is postulated to have proceeded by evolution in situ and not by additions and replacements from outside of the system.