There are two morphotypes of Penaeus semisulcatus described hitherto in the Persian Gulf, namely the banded and non-banded antennae morphotypes. In this study, we used morphometric measurements and two mitochondrial genes (16S rRNA and cytochrome oxidase subunit I-COI) to assess relationships between the two morphotypes of P. semisulcatus. Out of 25 morphological characters examined, 10 characters were found significantly different between the two morphotypes when tested against separate sexes or both sexes combined. Results from the 16S rRNA and COI sequence analysis of two morphotypes of P. semisulcatus morphotype showed up to 6% and 17% sequence divergence, respectively. The 16S rDNA and COI sequences of the non-banding morphotype were not only very different to those of the banding morphotype but was also very different to all other Penaeus species (i.e., P. monodon, P. merguiensis, and P. indicus) included in the study. Both parsimony and Neighbor-Joining trees based on 16S rDNA and COI sequences provide similar tree topology that clearly separated the two morphotypes into two distinct groups. Based on these findings, we propose the two morphotypes of P. semisulcatus to be relegated as two sympatric species.
The Blueline Rasbora (Rasbora sarawakensis) is a small ray-finned fish categorized under the genus Rasbora in the Cyprinidae family. In this study, the complete mitogenome sequence of R. sarawakensis was sequenced using four primers targeting overlapping regions. The mitogenome is 16,709 bp in size, accommodating 22 transfer RNA genes, 13 protein-coding genes, two ribosomal RNA genes and a putative control region. Identical gene organisation was detected between this species and other genus counterparts. The heavy strand houses 28 genes while the light strand stores the other nine genes. Most protein-coding genes employ ATG as start codon, excluding COI gene, which utilizes GTG instead. The central conserved sequence blocks (CSB-F, CSB-E and CSB-D), variable sequence blocks (CSB-3, CSB-2 and CSB-1) as well as the terminal associated sequence (TAS) are conserved in the control region. The maximum likelihood phylogenetic tree revealed the divergence of R. sarawakensis from the basal region of the Rasbora clade, where its evolutionary relationships with R. maculatus and R. pauciperforata are poorly resolved as indicated by the low bootstrap values. This work acts as steppingstone towards further molecular evolution and population genetics studies of Rasbora genus in future.
Vatica mangachapoi is a tree up to 20 m tall with white resinous. It is distributed in China (Hainan province), Indonesia, Malaysia (N Borneo), Philippines, Thailand, and Vietnam. It grows in forests on hills and mountain slopes below 700 metres. Its durable wood is used for making boats and building bridges and houses. It has been ranked as a VU (Vulnerable) species in China. Here we report and characterize the complete plastid genome sequence of V. mangachapoi in an effort to provide genomic resources useful for promoting its conservation and phylogenetic research. The complete plastome is 151,538 bp in length and contains the typical structure and gene content of angiosperm plastome, including two Inverted Repeat (IR) regions of 23,921 bp, a Large Single-Copy (LSC) region of 83,587 bp and a Small Single-Copy (SSC) region of 20,109 bp. The plastome contains 114 genes, consisting of 80 unique protein-coding genes, 30 unique tRNA gene, and 4 unique rRNA genes. The overall A/T content in the plastome of V. mangachapoi is 62.80%. The phylogenetic analysis indicated that V. mangachapoi and V. odorata is closely related and as an independent branch in Malvales in our study. The complete plastome sequence of V. mangachapoi will provide a useful resource for the conservation genetics of this species and for the phylogenetic studies for Vatica.
The increasing interest in understanding the evolutionary relationship between members of the Pteropodidae family has been greatly aided by genomic data from the Old World fruit bats. Here we present the complete mitogenome of Geoffroy's rousette, Rousettus amplexicaudatus found in Peninsular Malaysia . The mitogenome constructed is 16,511bp in length containing 37 genes; 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and a D-loop region. The overall base composition is estimated to be 32.28% for A, 25.64% for T, 14.09% for G and 27.98% for C, indicating a slightly AT rich feature (57.93%). A phylogenetic and BLASTn analysis against other available mitogenomes showed Malaysian R. amplexicaudatus matched 98% similarity to the same species in Cambodia and Vietnam. However, it differed considerably (92.53% similarity) with the same species in the Philippines. This suggests flexibility in Rousettus sp. with regards to adapting to mesic and dry habitats, ability for long-distance dispersal and remarkably precise lingual echolocation thus supporting its wide-range distribution and colonization. Further taxonomical and mitogenomic comparatives are required in resolving the evolutionary relationship between Rousettus spp.
Syzygium malaccense is native to Malaysia. It is sometimes called the malay apple, malay rose-apple, mountain rose-apple, mountain apple, water apple, or French cashew. The tree is very popular in many tropical and subtropical regions for its fruit and traditional medicine. The first complete chloroplast genome of Syzygium malaccense has been reported in this study. The complete chloroplast genome of Syzygium malaccense is 158,954 bp, composed of four regions: a large single-copy region with a size of 87,991 bp, a small single copy region with a size of 18,793 bp, and two inverted repeat regions with a size of 26,085 bp. The GC content is 36.97%. A total of 132 genes were annotated, including 84 encoding proteins, eight encoding rRNA genes, 37 encoding tRNA genes, and three encoding pseudo genes. Phylogenetic analysis showed that Syzygium aromaticum, Syzygium cumini, and Syzygium forrestii are closely related to Syzygium malaccense.
The polyhydroxyalkanoate (PHA) producing capability of four bacterial strains isolated from Antarctica was reported in a previous study. This study analyzed the PHA synthase genes and the PHA-associated gene clusters from the two antarctic Pseudomonas isolates (UMAB-08 and UMAB-40) and the two antarctic Janthinobacterium isolates (UMAB-56 and UMAB-60) through whole-genome sequence analysis. The Pseudomonas isolates were found to carry PHA synthase genes which fall into two different PHA gene clusters, namely Class I and Class II, which are involved in the biosynthesis of short-chain-length-PHA (SCL-PHA) and medium-chain-length-PHA (MCL-PHA), respectively. On the other hand, the Janthinobacterium isolates carry a Class I and an uncharacterized putative PHA synthase genes. No other gene involved in PHA synthesis was detected in close proximity to the uncharacterized putative PHA synthase gene in the Janthinobacterium isolates, therefore it falls into a separate clade from the ordinary Class I, II, III and IV clades of PHA synthase (PhaC) phylogenetic tree. Multiple sequence alignment showed that the uncharacterized putative PHA synthase gene contains all the highly conserved amino acid residues and the proposed catalytic triad of PHA synthase. PHA biosynthesis and in vitro PhaC enzymatic assay results showed that this uncharacterized putative PHA synthase from Janthinobacterium sp. UMAB-60 is funtional. This report adds new knowledge to the PHA synthase database as we describe scarce information of PHA synthase genes and PHA-associated gene clusters from the antarctic bacterial isolates (extreme and geographically isolated environment) and comparing with those from non-antarctic PHA-producing bacteria.
The estuarine firefly, Pteroptyx tener, aggregates in the thousands in mangrove trees lining tidal rivers in Southeast Asia where they engage one another in a nocturnal, pre-mating ritual of synchronised courtship flashes. Unfortunately, populations of the species by virtue of being restricted to isolated estuarine rivers systems in the region, are at risk of genetic isolation. Because of this concern we undertook the task of sequencing and characterising the mitochondrial DNA genome of P. tener, as the first step towards helping us to characterise and better understand their genetic diversity. We sequenced and assembled the mitochondrial DNA genome of P. tener from two male and female specimens from the district of Kuala Selangor in Peninsular Malaysia and announce the molecules in this publication. We also reconstructed the phylogenetic trees of all available lampyrids mitogenomes and suggest the need to re-examine our current understanding of their classification which have largely been based on morphological data and the cox1 gene. Separately, our analysis of codon usage patterns among lampyrid mitogenomes showed that the codon usage in a majority of the protein-coding genes were non-neutral. Codon usage patterns between mitogenome sequences of P. tener were, however, largely neutral. Our findings demonstrate the usefulness of mitochondrial genes/mitogenomes for analysing both inter- and intra- specific variation in the Lampyridae to aid in species discovery in this highly variable genus; and elucidate the phylogenetic relationships of Pteroptyx spp. from the region.
Bacteria of the genus Komagataeibacter are described to be the most noteworthy for having several of its species being efficient and strong cellulose producers. The 16S ribosomal RNA (rRNA) gene analysis is often used for the identification and taxonomic classification of these bacteria species. In order to observe the phylogenetic relationship among Komagataeibacter sp., twelve sequences of the 16S rRNA gene with three sequences each for species namely Komagataeibacter europaeus, Komagataeibacter hansenii, Komagataeibacter intermedius and Komagataeibacter xylinus were retrieved from NCBI GenBank database. The sequences were aligned and analysed using PAUP, OrthoANI and BLAST, followed by the phylogenetic tree construction using a Maximum Likelihood method. The parsimony character diagnostic analysis showed very few numbers of parsimony- informative characters present in the aligned sequences which is only 1.5% of the total characters. The inferred phylogenetic relationships demonstrated the unexpected positioning of K. xylinus (GQ240638: Gluconacetobacter xylinus strain) and K. xylinus (KC11853: G. xylinus strain) into the clades of K. europaeus and K. hansenii respectively. The also very low bootstrap values of the branch points linking the K. europaeus species indicated low support for the produced topologies. The findings of this study indicate that more phylogenies information can be attained by increasing the taxon sampling. In addition, more robust molecular data are needed to infer the phylogenetic relationships between the Komagataeibacter species more accurately.
The Christmas Island red crab, Gecarcoidea natalis, is an herbivorous land crab that consumes mostly fallen leaf litter. In order to subsist, G. natalis would need to have developed specialised digestive enzymes capable of supplying significant amounts of metabolisable sugars from this diet. To gain insights into the carbohydrate metabolism of G. natalis, a transcriptome assembly was performed, with a specific focus on identifying transcripts coding for carbohydrate active enzyme (CAZy) using in silico approaches. Transcriptome sequencing of the midgut gland identified 70 CAZy-coding transcripts with varying expression values. At least three newly discovered putative GH9 endo-β-1,4-glucanase ("classic cellulase") transcripts were highly expressed in the midgut gland in addition to the previously characterised GH9 and GH16 (β-1,3-glucanase) transcripts, and underscoring the utility of whole transcriptome in uncovering new CAZy-coding transcripts. A highly expressed transcript coding for GH5_10 previously missed by conventional screening of cellulase activity was inferred to be a novel endo-β-1,4-mannase in G. natalis with in silico support from homology modelling and amino acid alignment with other functionally validated GH5_10 proteins. Maximum likelihood tree reconstruction of the GH5_10 proteins demonstrates the phylogenetic affiliation of the G. natalis GH5_10 transcript to that of other decapods, supporting endogenous expression. Surprisingly, crustacean-derived GH5_10 transcripts were near absent in the current CAZy database and yet mining of the transcriptome shotgun assembly (TSA) recovered more than 100 crustacean GH5_10s in addition to several other biotechnological relevant CAZys, underscoring the unappreciated potential of the TSA database as a valuable resource for crustacean CAZys.
During the study into the microbial biodiversity and bioactivity of the Microcystis phycosphere, a new yellow-pigmented, non-motile, rod-shaped bacterium containing polyhydroxybutyrate granules designated as strain Z10-6T was isolated from highly-toxic Microcystis aeruginosa Kützing M.TN-2. The new isolate produces active bioflocculating exopolysaccharides. Phylogenetic analysis based on 16S rRNA gene sequences indicated strain Z10-6T belongs to the genus Sphingopyxis with highest similarity to Sphingopyxis solisilvae R366T (98.86%), and the similarity to other Sphingopyxis members was less than 98.65%. However, both low values obtained by phylogenomic calculation of average nucleotide identity (ANI, 85.5%) and digital DNA-DNA hybridization (dDDH, 29.8%) separated the new species from its closest relative. The main polar lipids were sphingoglycolipid, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unidentified glycolipid and one unidentified aminophospholipid. The predominant fatty acids were summed feature 8, C17:1ω6c, summed feature 3, C16:0, C18:1ω7c 11-methyl and C14:0 2-OH. The respiratory quinone was ubiqunone-10, with spermidine as the major polyamine. The genomic DNA G + C content was 64.8 mol%. Several biosynthesis pathways encoding for potential new bacterial bioactive metabolites were found in the genome of strain Z10-6T. The polyphasic analyses clearly distinguished strain Z10-6T from its closest phylogenetic neighbors. Thus, it represents a novel species of the genus Sphingopyxis, for which the name Sphingopyxis microcysteis sp. nov. is proposed. The type strain is Z10-6T (= CCTCC AB2017276T = KCTC 62492T).
Marine phycosphere harbors unique cross-kingdom associations with enormous ecological significance in aquatic ecosystems as well as relevance for algal biotechnology industry. During our investigating the microbial composition and bioactivity of marine phycosphere microbiota (PM), a novel lightly yellowish and versatile bacterium designated strain AM1-D1T was isolated from cultivable PM of marine dinoflagellate Alexandrium minutum amtk4 that produces high levels of paralytic shellfish poisoning toxins (PSTs). Strain AM1-D1T demonstrates notable bioflocculanting bioactivity with bacterial exopolysaccharides (EPS), and microalgae growth-promoting (MGP) potential toward its algal host. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain AM1-D1T was affiliated to the members of genus Sulfitobacter within the family Rhodobacteraceae, showing the highest sequence similarity of 97.9% with Sulfitobacter noctilucae NB-68T, and below 97.8% with other type strains. The complete genome of strain AM1-D1T consisted of a circular 3.84-Mb chromosome and five circular plasmids (185, 95, 15, 205 and 348 Kb, respectively) with the G+C content of 64.6%. Low values obtained by phylogenomic calculations on the average nucleotide identity (ANI, 77.2%), average amino acid identity (AAI, 74.7%) and digital DNA-DNA hybridization (dDDH, 18.6%) unequivocally separated strain AM1-D1T from its closest relative. The main polar lipids were identified as phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, diphosphatidylglycerol, one unidentified phospholipid and one unidentified lipid. The predominant fatty acids (> 10%) were C18:1 ω7c, C19:0 cyclo ω8c and C16:0. The respiratory quinone was Q-10. The genome of strain AM1-D1T was predicted to encode series of gene clusters responsible for sulfur oxidation (sox) and utilization of dissolved organic sulfur exometabolites from marine dinoflagellates, taurine (tau) and dimethylsulfoniopropionate (DMSP) (dmd), as well as supplementary vitamin B12 (cob), photosynthesis carotenoids (crt) which are pivotal components during algae-bacteria interactions. Based on the evidences by the polyphasic characterizations, strain AM1-D1T represents a novel species of the genus Sulfitobacter, for which the name Sulfitobacter alexandrii sp. nov. is proposed. The type strain is AM1-D1T (= CCTCC 2017277T = KCTC 62491T).
The subspecies classification of Mycobacteroides abscessus complex into M. abscessus, M. massiliense and M. bolletii requires the amplification and sequencing of multiple genes. The objective of this study was to evaluate the possibility of subspecies classification using a single PCR target. An in silico study was performed to classify 1613 strains deposited in a public database using 9 genes (partial gene sequences of hsp65, rpoB, sodA, argH, cya, glpK, gnd, and murC, and the full gene sequence of MAB_3542c). We found the housekeeping gene gnd to be able to classify the M. abscessus subspecies with high accuracy (99.94%). A single-gene PCR approach based on gnd would be a suitable replacement for the more expensive, labor-intensive and time-consuming multi-gene PCR analysis currently in use for the subspecies identification of M. abscessus.
Actinobacteria from underexplored and unusual environments have gained significant attention for their capability in producing novel bioactive molecules of diverse chemical entities. Streptomyces is the most prolific Actinobacteria in producing useful molecules. Rapid decline effectiveness of existing antibiotics in the treatment of infections are caused by the emergence of multidrug-resistant pathogens. Intensive efforts are urgently required in isolating non-Streptomyces or rare Actinobacteria and understanding of their distribution in the harsh environment for new drug discovery. In this study, pretreatment of soil samples with 1.5% phenol was used for the selective isolation of Actinobacteria from Dee Island and Greenwich Island. A high number of non-Streptomyces (69.4%) or rare Actinobacteria was significantly recovered despite the Streptomyces (30.6%), including the genera Micromonospora, Micrococcus, Kocuria, Dermacoccus, Brachybacterium, Brevibacterium, Rhodococcus, Microbacterium and Rothia. Reduced diversity and shift of distribution were observed at the elevated level of soil pH. The members of genera Streptomyces, Micromonospora and Micrococcus were found to distribute and tolerate to a relatively high pH level of soil (pH 9.4-9.5), and could potentially be alkaliphilic Actinobacteria. The phylogenetic analysis had revealed some potentially new taxa members of the genera Micromonospora, Micrococcus and Rhodococcus. Principal Component Analysis of soil samples was used to uncover the factors that underlie the diversity of culturable Actinobacteria. Water availability in soil was examined as the principal factor that shaped the diversity of the Actinobacteria, by providing a dynamic source for microbial interactions and elevated diversity of Actinobacteria.
Most new cryptic species are described using conventional tree- and distance-based species delimitation methods (SDMs), which rely on phylogenetic arrangements and measures of genetic divergence. However, although numerous factors such as population structure and gene flow are known to confound phylogenetic inference and species delimitation, the influence of these processes is not frequently evaluated. Using large numbers of exons, introns, and ultraconserved elements obtained using the FrogCap sequence-capture protocol, we compared conventional SDMs with more robust genomic analyses that assess population structure and gene flow to characterize species boundaries in a Southeast Asian frog complex (Pulchrana picturata). Our results showed that gene flow and introgression can produce phylogenetic patterns and levels of divergence that resemble distinct species (up to 10% divergence in mitochondrial DNA). Hybrid populations were inferred as independent (singleton) clades that were highly divergent from adjacent populations (7%-10%) and unusually similar (<3%) to allopatric populations. Such anomalous patterns are not uncommon in Southeast Asian amphibians, which brings into question whether the high levels of cryptic diversity observed in other amphibian groups reflect distinct cryptic species-or, instead, highly admixed and structured metapopulation lineages. Our results also provide an alternative explanation to the conundrum of divergent (sometimes nonsister) sympatric lineages-a pattern that has been celebrated as indicative of true cryptic speciation. Based on these findings, we recommend that species delimitation of continuously distributed "cryptic" groups should not rely solely on conventional SDMs, but should necessarily examine population structure and gene flow to avoid taxonomic inflation.
Most female black flies in the genus Simulium are blood-sucking flies and they can cause various parasitic diseases in human and animal. A total of 94 species of black flies have been reported in Malaysia, however, their biting behavior and role as vector of infectious agents remain understudied. To fill in this knowledge gap, we attempted to survey adult black flies from field populations in Peninsular Malaysia. In a survey carried out in 2017 at Tasik Kenyir, Terengganu, three females were caught while attracted and landed on human skin. Further morphological and molecular analyses showed that the specimens were identical to Simulium (Gomphostilbia) aziruni Takaoka, Hashim & Chen of the Simulium gombakense species-group. This is the first report on a black fly species attracted to human in Malaysia which serves as a steppingstone towards in-depth studies for black flies in this region.
Lizards and snakes (squamates) are known for their varied sex determining systems, and gecko lizards are especially diverse, having evolved sex chromosomes independently multiple times. While sex chromosomes frequently turnover among gecko genera, intrageneric turnovers are known only from Gekko and Hemidactylus. Here, we used RADseq to identify sex-specific markers in two species of Burmese bent-toed geckos. We uncovered XX/XY sex chromosomes in Cyrtodactylus chaunghanakwaensis and ZZ/ZW sex chromosomes in Cyrtodactylus pharbaungensis. This is the third instance of intrageneric turnover of sex chromosomes in geckos. Additionally, Cyrtodactylus are closely related to another genus with intrageneric turnover, Hemidactylus. Together, these data suggest that sex chromosome turnover may be common in this clade, setting them apart as exceptionally diverse in a group already known for diverse sex determination systems.
Staphylococcus kloosii, an orange pigment-producing bacterium, was isolated from the respiratory tree of Holothuria (Mertensiothuria) leucospilota (Brandt 1835) from Teluk Nipah, Pangkor Island, Perak, Malaysia. This report is the first documentation of this Gram-positive strain, referred to as Strain 68 in Malaysia. A partial 16S ribosomal RNA gene sequence of the mesophilic strain has been registered with GenBank (National Center for Biotechnology Information, US National Library of Medicine) with accession number JX102547. Phylogenetic analysis using the neighbour-joining method further supported the identification of Strain 68 as S. kloosii. The circular strain produced orange pigments on tryptone glucose yeast extract agar (TGYEA) and in nutrient broth (NB) at approximately pH 7. The visible spectra of ethanolic and methanolic pigment extracts of the bacterial strain were considered identical with λmax at 426, 447 and 475 nm and λmax at 426, 445 and 473 nm, respectively. Both visible spectra resemble the visible spectra of lutein, which is a commercial carotenoid; however, further analyses are required to confirm the identity of this pigment. The methanolic extracts of the intracellular pigments comprised at least three pigment compounds: an orange pigment compound (major compound), a yellow pigment compound (the least polar) and a pink pigment compound (the most polar). These findings are the first documentation of the pigment composition of S. kloosii as no such record could be found to date.
Different clades belonging to the cosmopolitan marine diatom Pseudo-nitzschia pungens appear to be present in different oceanic environments, however, a 'hybrid zone', where populations of different clades interbreed, has also been reported. Many studies have investigated the sexual reproduction of P. pungens, focused on morphology and life cycle, rather than the role of sexual reproduction in mixing the genomes of their parents. We carried out crossing experiments to determine the sexual compatibility/incompatibility between different clades of P. pungens, and examined the genetic polymorphism in the ITS2 region. Sexual reproduction did not occur only between clades II and III under any of experimental temperature conditions. Four offspring strains were established between clade I and III successfully. Strains established from offspring were found interbreed with other offspring strains as well as viable with their parental strains. We confirmed the hybrid sequence patterns between clades I and III and found novel sequence types including polymorphic single nucleotide polymorphisms (SNPs) in the offspring strains. Our results implicate that gene exchange and mixing between different clades are still possible, and that sexual reproduction is a significant ecological strategy to maintain the genetic diversity within this diatom species.
Avian Infectious Bronchitis (IB) is a highly contagious disease which can cause huge economic losses to the poultry industry. Forty five IB viruses (IBV) were isolated from poultry in Malaysia during 2014-2016. Phylogenetic analysis of the spike glycoprotein 1 (S1) gene revealed that all isolates were clustered into five distinct groups. The predominant type of IBV isolated was QX strains (47%), second was 4/91 type (27%), followed by Malaysian strain MH5365/95 (13%), Massachusetts type (11%) and finally Taiwanese strains (2%). Four types of S1 protein cleavage recognition motifs were found among the isolates which includes HRRRR, RRSRR, RRFRR and RRVRR. To our knowledge, this is the first report describing the motif RRVRR and are unique to Malaysian strains. Six IBVs were grouped in Malaysian MH5365/95 strains. Among these, one isolate was different from others where it only shared 82% identity with MH5365/95 and to others. It formed its own branch in the Malaysian cluster suggesting it may be a variant unique to Malaysia. Alignment analysis of the S1 amino acid sequences indicated that point mutations, insertions and deletions contribute to the divergence of IB variants. This study indicated at least five groups of IBV are circulating in Malaysia with most of the isolates belonged to QX strains. As new IBV variants continue to emerge, further study need to be carried out to determine whether the current available vaccine is able to give protection against the circulating virus.
Background: The tissue specimens used for extraction of DNA in this study were from rodents trapped in four states in Peninsular Malaysia, namely Kedah, Kelantan, Selangor and Johor. Methods: Histological sections of these rodent muscle tissues stained with hematoxylin and eos in showed infection with Sarcocystis spp. Based on these results, the current study was carried out to determine the phylogenetic relationship among the identified Sarcocystis spp. in these rodents.The formalin fixed paraffin embedded (FFPE) rodent muscle blocks were subjected to DNA extraction and followed with semi nested PCR targeting 5’ and 3’ regions of 18S rRNA of Sarcocystis spp. Results: Phylogenetic analysis showed two distinct groups of Sarcocystis spp. among the rodents in Peninsular Malaysia. Most of the identified Sarcocystis spp. were genetically closely related to Sarcocystis rodentifelis and Sarcocystis muris and were also observed to be genetically closely related to Sarcocystis sp. ex Columba livia and Sarcocystis sp. cyst type I ex Anser albifrons. Conclusion: Further classification to confirm these Sarcocystis spp. was not possible as only partial sequences of 18S rRNA was available and this was insufficient for
optimal differentiation.