Plant DNA barcodes and assessment of phylogenetic community structure of a tropical mixed dipterocarp forest in Brunei Darussalam (Borneo)

DNA barcoding is a fast and reliable tool to assess and monitor biodiversity and, via community phylogenetics, to investigate ecological and evolutionary processes that may be responsible for the community structure of forests. In this study, DNA barcodes for the two widely used plastid coding regions rbcL and matK are used to contribute to identification of morphologically undetermined individuals, as well as to investigate phylogenetic structure of tree communities in 70 subplots (10 × 10m) of a 25-ha forest-dynamics plot in Brunei (Borneo, Southeast Asia). The combined matrix (rbcL + matK) comprised 555 haplotypes (from ≥154 genera, 68 families and 25 orders sensu APG, Angiosperm Phylogeny Group, 2016), making a substantial contribution to tree barcode sequences from Southeast Asia. Barcode sequences were used to reconstruct phylogenetic relationships using maximum likelihood, both with and without constraining the topology of taxonomic orders to match that proposed by the Angiosperm Phylogeny Group. A third phylogenetic tree was reconstructed using the program Phylomatic to investigate the influence of phylogenetic resolution on results. Detection of non-random patterns of community assembly was determined by net relatedness index (NRI) and nearest taxon index (NTI). In most cases, community assembly was either random or phylogenetically clustered, which likely indicates the importance to community structure of habitat filtering based on phylogenetically correlated traits in determining community structure. Different phylogenetic trees gave similar overall results, but the Phylomatic tree produced greater variation across plots for NRI and NTI values, presumably due to noise introduced by using an unresolved phylogenetic tree. Our results suggest that using a DNA barcode tree has benefits over the traditionally used Phylomatic approach by increasing precision and accuracy and allowing the incorporation of taxonomically unidentified individuals into analyses

Phylogenetic community structure assessment of a mixed Dipterocarp forest using DNA barcoding and molecular phylogeny of the dominant tree family Dipterocarpaceae

Für das Barcoding von Pflanzen werden standardmäßig zwei Marker verwendet: Ribulose-1,5-bisphosphat-Carboxylase/Oxygenase (rbcL) und Maturase K (matK). Im Gegensatz zu der weitestgehend konservierten rbcL-Barcoding-Region ist die matK-Barcoding-Region sehr variable (ca. dreimal so variable wie rbcL), was mit Schwierigkeiten der Auswahl der Primer, welche für die Vervielfältigung der DNS während der Polymerase-Ketten-Reaktion (PCR) benötigt werden, einhergeht. Dies ist insbesondere der Fall, wenn ein weites Spektrum an verschiedenen Pflanzenfamilien untersucht wird. Frühere Studien verwendeten jeweils unterschiedliche Primerkombinationen für verschiedene Familien. Dieses Verfahren ist jedoch zeitaufwendig und teuer. In der ersten Studie dieser Arbeit, wurden 14 universelle matK-Primer, unter Verwendung von kompletten matK-Sequenzen aus GenBank, entwickelt. Mittels Kombination dieser Primer in einer Multiplex-PCR konnte die matK-Barcoding-Region aller Angiospermen erfolgreich amplifiziert werden. Die matK-Barcoding-Region wurde, zusammen mit der rbcL-Barcoding-Region, in einer zweiten Studie verwendet, um Bäume und Sträucher (3237 Individuen) in mehreren Quadraten eines 25 ha Dipterocarpaceaen-Mischwaldes in Brunei Darussalam (Borneo, Südostasien) meist auf Gattungsebene zu identifizieren und die phylogenetische Gemeinschaftsstruktur dieses Waldes zu beurteilen. Die kombinierte Matrix aus rbcL- und matK-Barcoding-Regionen [555 Haplotypen, die zu ≥154 Gattungen, 68 Familien, 25 Ordnungen nach der Angiosperm Phylogeny Group (APG) gehören], wurde verwendet, um phylogenetische Verwandtschaftsbeziehungen zu erstellen [mit und ohne Verwendung eines APG-Baums auf Ordnungsebene (APGIII)]. Ein dritter phylogenetischer Baum wurde unter Verwendung des Programms Phylomatic rekonstruiert. Dieses Programm wird traditionell von vielen Ökologen verwendet und reduziert einen existierenden Referenzbaum so, dass nur Taxa, welche in der betreffenden Pflanzengesellschaft vorkommen, enthalten sind. Die verschiedenen phylogenetischen Bäume wurden verwendet um Gesellschaftsindizes [Net Relatedness Index (NRI) and Nearest Taxon Index (NTI)] zu berechnen. Die Gesellschaftsindizes deckten jeweils die gleichen Formen der Gesellschaftsstruktur auf: in den meisten Fällen zufällige Verteilung oder phylogenetische Gruppierung. Phylogenetische Gruppierung weist auf den möglichen Einfluss abiotischer Faktoren auf die Gesellschaftsstruktur des Waldes hin. Allerdings wiesen die Gesellschaftsindizes unter Verwendung des Phylomatic-Baumes eine deutlich höhere Variation auf, welche sich mit statistischem Rauschen, einhergehend mit der niedrigen Auflösung dieses phylogenetischen Baumes, erklären lässt. Weiterhin beschäftigt sich diese Arbeit mit der molekularen Phylogenie der ökologisch und ökonomisch wichtigen Familie der Flügelfluchtgewächse (Dipterocarpaceae, Malvales), welche die dominierenden Bäume im untersuchten Wald darstellen. Die Familie der Flügelfruchtgewächse wird in drei Unterfamilien gegliedert: Dipterocarpoideae, die größte, vorwiegend in Asien vorkommende Unterfamilie, Monotoideae in Afrika, Madagaskar und dem kolumbianischen Amazonas und Pakaraimaeoideae aus den Hochländern Guayanas und Venezuela. Datierungsanalysen, basierend auf DNA-Sequenzdaten zeigen, dass die Dipterocarpoideae ca. 55 Millionen Jahre alt sind. Phylogenetische Analyse verschiedener Plastidenregionen aller drei Unterfamilien, sowie Vertreter der nah verwandten Familien Sarcolaenaceae, Cistaceae und Bixaceae zeigt verwandtschaftliche Unterschiede zu jenen in bisherigen morphologischen Klassifikationen vorgeschlagenen. Pakaraimaea (Unterfamilie Pakaraimaeoideae) bildet eine Gruppe mit Cistaceaea. Monotoideae bildet eine schwach unterstützte Schwestergruppe zu Dipterocarpoideae. In Bezug auf die Unterfamilie Dipterocarpoideae, ist Dipterocarpus Schwestergruppe zu Dryobalanops und Shoreeae, was dem morphologischen Konzepte der beiden Tribus Dipterocarpeae (Anisoptera, Cotylelobium, Dipterocarpus, Stemonoporus, Vatica, Vateria und Vateriopsis) und Shoreeae (Hopea, Parashorea, Neobalanocarpus und Shorea) im Sinne von Ashton widerspricht. Weiterhin ist die Gattung Shorea (sensu Ashton) nicht monophyletisch. Die untersuchten Genomgrößen sind klein (0,3264-0,6724 pg). Neue Chromosomenzahlen wurden ermittelt (Dipterocarpus zeylanicus Thwaites: 2n = 22; Shorea megistophylla P.S.Ashton: 2n = 14; Hopea jucunda Thwaites: 2n = 21; Shorea oblongifolia Thwaites: 2n = 14; and Vatica endertii Slooten: 2n = 22). Diese entsprechen früheren Aufzeichnungen in der Familie Dipterocarpaceae. RAD-Sequenzierung (Restriction Site Associated DNA Sequencing, eine Next-Generation-Sequenziermethode) wurde erfolgreich verwendet, um phylogenetische Verwandtschaftsbeziehungen innerhalb der Shoreeae aufzulösen. Analysen von Tausenden von RAD-abgeleiteten SNPs (Single Nucleotid Polymorphisms) führen zu kongruenten, aber viel besser aufgelösten Bäumen im Vergleich zu jenen, die aus Sanger-Sequenzierung von Plastidenregionen resultieren. Bei der polyphyletischen Gattung Shorea wird auch die Monophylie einiger (Unter-)Sektionen nach Ashton nicht gestützt, während Gruppierungen nach Maury unterstützt werden. Dies trifft ebenfalls für einige (Unter-)Sektionen der monophyletischen Gattung Hopea zu. Einblicke in die Evolution von Blütenmerkmalen stützen die auf Bestäubungsbiologie und Biogeographie basierende Hypothese, dass Blüten mit großen, länglichen Antheren und mehr als 15 Staubblätternmit kurzen Anhängen einen plesiomorphen Merkmalszustand in der Unterfamilie Dipterocarpoideae darzustellen scheinen.For DNA barcoding, the two markers ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) and maturase K (matK) are widely used in plants. In contrast to the well conserved rbcL barcode region, the matk is rapidly evolving and therefore highly variable, approx. three times more variable than that of rbcL. PCR amplification of matK is often difficult especially when dealing with many different plant families. Earlier DNA barcoding studies have used different primer pair combinations for different plant families which is time consuming and costly. In this project, 14 universal matk primers were developed using complete matk sequences available from GenBank. These primers, when combined in a multiplex PCR, amplify the target region across a wide range of Angiosperms families. In this study, the matK, together with the rbcL barcode region, successfully identified trees and shrubs (3237 individuals) mostly at generic level, which helped to assess the phylogenetic community structure in several subplots of a 25 ha mixed Dipterocarp forest in Brunei Darussalam (Borneo, Southeast Asia). The combined matrix of rbcL + matK barcodes [555 haplotypes which belong to ≥154 genera, 68 families, 25 orders sensu Angiosperm Phylogeny Group (APG)] was used to reconstruct phylogenetic relationships, with and without constraining the topology of taxonomic orders to match that proposed by the Angiosperm Phylogeny Group (APG III). A third phylogenetic tree was reconstructed using the program Phylomatic which trims a reference tree to taxa occurring in the community. This program is traditionally used by many ecologists. The different phylogenetic trees obtained were used to calculate community metrics [net relatedness index (NRI) and nearest taxon index (NTI)]. The community indices detected the same patterns of community structure: in most cases it was either random community assembly or phylogenetically clustering, irrespective of the different phylogenetic trees used for calculations. Phylogenetic clustering indicates that habitat filtering plays a role in assembly processes. However, the Phylomatic tree produces greater variation across the plots for NRI and NTI, presumably due to noise introduced by using an unresolved phylogenetic tree. Dipterocarpaceae (Malvales) are the dominant trees in the study plot, which led to investigation of the molecular phylogeny of the whole family including all subfamilies. Dipterocarpaceae comprises three subfamilies: the largest Asian subfamily Dipterocarpoideae, Monotoideae from Africa, Madagascar and the Colombian Amazon, and Pakaraimaeoideae from Guaianan Highlands and Venezuela. Molecular clock analysis reveals that extant Dipterocarpoideae diverged approx. 55 Mya. Phylogenetic analysis of plastid regions including all three subfamilies as well as representatives of closely related families Sarcolaenaceae, Cistaceae, and Bixaceae, highlights differences from the previous morphological classifications. Pakaraimaea of the monotypic subfamily Pakaraimaeoideae is assigned to Cistaceae. Monotoideae is weakly supported as sister to Dipterocarpoideae. In the subfamily Dipterocarpoideae, Dipterocarpus is sister to Dryobalanops and tribe Shoreeae which contradicts the morphological concepts of the tribes Dipterocarpeae (Anisoptera, Cotylelobium, Dipterocarpus, Stemonoporus, Vatica, Vateria, and Vateriopsis) and Shoreeae (Hopea, Parashorea, Neobalanocarpus und Shorea) sensu Ashton. Further, the genus Shorea (sensu Ashton) is not monophyletic. Genome sizes of the species examined are small (0.3264–0.6724 pg). New chromosome numbers are reported (Dipterocarpus zeylanicus Thwaites: 2n = 22; Shorea megistophylla P.S.Ashton: 2n = 14; Hopea jucunda Thwaites: 2n = 21; Shorea oblongifolia Thwaites: 2n = 14; and Vatica endertii Slooten: 2n = 22). These correspond to earlier records in the family Dipterocarpaceae. RADseq (restriction site associated DNA sequencing; next generation sequencing) was successfully used to infer species relationships within the tribe Shoreeae. Analyses of thousands of RAD derived SNPs lead to congruent and much better resolved trees compared to those obtained by sanger sequencing of plastid regions. Regarding the polyphyletic genus Shorea, taxonomic grouping based on wood anatomy is supported by RADseq, but it contradicts some (sub-)sectional relationships proposed by Ashton. This was also the case for some (sub-)sections of the genus Hopea. Insights into the evolution of floral traits support the hypothesis that flowers with large, oblong anthers with short appendages and more than 15 stamens are plesiomorphic characters in the subfamily Dipterocarpoideae, as hypothesised previously based on pollination biology and biogeography

Isolation, characterization and cross amplification of eleven novel microsatellite loci for the hydrozoan coral Millepora

Milleporidae are of high ecological and economic importance, as, together with the scleractinian corals, they belong to the main reef builders of tropical coral reefs. Coral reefs face severe threats mainly due to anthropogenic disturbance. Understanding their population structure and dynamics is crucial for any conservation effort. Here we report the first microsatellite loci for the Milleporidae. Eleven polymorphic markers were developed for the hydrozoan corals Millepora dichotoma from the Great Barrier Reef (Australia) and tested for amplification in M. dichotoma from the Red Sea (Egypt), as well as for Millepora platyphylla from the Pacific Ocean (Moorea, French Polynesia). All loci were variable with 4–15 alleles per locus. Nine loci were transferable between geographic regions and species. These are the first microsatellites for hydrozoan corals. They will provide valuable tools for characterizing the population structure and genetic diversity of the group thereby benefitting coral reef conservation

Holotype sequencing of Silvatares holzenthali (Trichoptera, Pisuliidae)

While “DNA barcodes” are often provided, the whole mitochondrial and nuclear genome are rarely considered to be included in species descriptions. This is unfortunate because whole genome sequencing of holotypes allows eternal genetic characterization of the most representative specimen for a given species. Thus, de novo genomes are invaluable added resources and important additional diagnostic characters in species descriptions, provided the integrity of the holotype specimens remains intact. Here, we used a minimally invasive method to extract DNA of the type specimen of the recently described caddisfly species Silvatares holzenthali (Trichoptera, Pisuliidae) from the Democratic Republic of the Congo. A low-cost next generation sequencing strategy was used to generate the complete mitochondrial and draft nuclear genome of the holotype. The data in its current form is an important extension to the morphological species description and valuable for phylogenomic studies

Conservation of Three-Dimensional Structure of Lepidoptera and Trichoptera L-Fibroins for 290 Million Years

The divergence of sister orders Trichoptera (caddisflies) and Lepidoptera (moths and butterflies) from a silk-spinning ancestor occurred around 290 million years ago. Trichoptera larvae are mainly aquatic, and Lepidoptera larvae are almost entirely terrestrial—distinct habitats that required molecular adaptation of their silk for deployment in water and air, respectively. The major protein components of their silks are heavy chain and light chain fibroins. In an effort to identify molecular changes in L-fibroins that may have contributed to the divergent use of silk in water and air, we used the ColabFold implementation of AlphaFold2 to predict three-dimensional structures of L-fibroins from both orders. A comparison of the structures revealed that despite the ancient divergence, profoundly different habitats, and low sequence conservation, a novel 10-helix core structure was strongly conserved in L-fibroins from both orders. Previously known intra- and intermolecular disulfide linkages were accurately predicted. Structural variations outside of the core may represent molecular changes that contributed to the evolution of insect silks adapted to water or air. The distributions of electrostatic potential, for example, were not conserved and present distinct order-specific surfaces for potential interactions with or modulation by external factors. Additionally, the interactions of L-fibroins with the H-fibroin C-termini are different for these orders; lepidopteran L-fibroins have N-terminal insertions that are not present in trichopteran L-fibroins, which form an unstructured ribbon in isolation but become part of an intermolecular β-sheet when folded with their corresponding H-fibroin C-termini. The results are an example of protein structure prediction from deep sequence data of understudied proteins made possible by AlphaFold2

Holotype sequencing of Silvatares holzenthali Rázuri-Gonzales, Ngera & Pauls, 2022 (Trichoptera, Pisuliidae)

While DNA barcodes are increasingly provided in descriptions of new species, the whole mitochondrial and nuclear genomes are still rarely included. This is unfortunate because whole genome sequencing of holotypes allows perpetual genetic characterization of the most representative specimen for a given species. Thus, de novo genomes are invaluable additional diagnostic characters in species descriptions, provided the structural integrity of the holotype specimens remains intact. Here, we used a minimally invasive method to extract DNA of the type specimen of the recently described caddisfly species Silvatares holzenthali Rázuri-Gonzales, Ngera & Pauls, 2022 (Trichoptera: Pisuliidae) from the Democratic Republic of the Congo. A low-cost next generation sequencing strategy was used to generate the complete mitochondrial and draft nuclear genome of the holotype. The data in its current form is an important extension to the morphological species description and valuable for phylogenomic studies

Molecular phylogeny helps to delimit <i>Plectranthus</i> <i>hadiensis</i> from its related morph occurring in Sri Lanka

Plectranthus hadiensis is an important medicinal plant in Sri Lanka. It was considered a separate species, P. zeylanicus, endemic to the island until its inclusion, as P. hadiensis var. tomentosus, together with morphs from southern Africa in the revised species concept of P. hadiensis. However, there are morphological, chemical, and therapeutic differences between the African and Sri Lankan morphs. We used eight molecular markers in a phylogenetic study to clarify the species concept of P. hadiensis and to investigate whether it should include the Sri Lankan morph. We examined the position of the two P. hadiensis morphs in relation to eight other Plectranthus species. The maximum likelihood tree revealed three clades: a weakly supported clade including P. calycinus, P. glabratus, P. fruticosus, and P. malabaricus; a highly supported clade including P. amboinicus and African and Sri Lankan specimens of P. hadiensis; and a highly supported clade formed by P. barbatus, P. caninus, and P. hadiensis var. tomentosus. The African P. hadiensis specimens form a highly supported subclade sister to a subclade containing the Sri Lankan P. hadiensis, suggesting that the subclades correspond to either two sister species or two subspecies. We propose that they are more likely to be sister species given the differences in morphology, chemistry, and chromosome number

Analyses of 600+ insect genomes reveal repetitive element dynamics and highlight biodiversity-scale repeat annotation challenges.

Repetitive elements (REs) are integral to the composition, structure, and function of eukaryotic genomes, yet remain understudied in most taxonomic groups. We investigated REs across 601 insect species and report wide variation in RE dynamics across groups. Analysis of associations between REs and protein-coding genes revealed dynamic evolution at the interface between REs and coding regions across insects, including notably elevated RE-gene associations in lineages with abundant long interspersed nuclear elements (LINEs). We leveraged this large, empirical data set to quantify impacts of long-read technology on RE detection and investigate fundamental challenges to RE annotation in diverse groups. In long-read assemblies, we detected ∼36% more REs than short-read assemblies, with long terminal repeats (LTRs) showing 162% increased detection, whereas DNA transposons and LINEs showed less respective technology-related bias. In most insect lineages, 25%-85% of repetitive sequences were unclassified following automated annotation, compared with only ∼13% in Drosophila species. Although the diversity of available insect genomes has rapidly expanded, we show the rate of community contributions to RE databases has not kept pace, preventing efficient annotation and high-resolution study of REs in most groups. We highlight the tremendous opportunity and need for the biodiversity genomics field to embrace REs and suggest collective steps for making progress toward this goal

Long-reads are revolutionizing 20 years of insect genome sequencing

The first insect genome assembly (Drosophila melanogaster) was published two decades ago. Today, nuclear genome assemblies are available for a staggering 601 insect species representing 20 orders. In this study, we analyzed the most-contiguous assembly for each species and provide a "state-of-the-field" perspective, emphasizing taxonomic representation, assembly quality, gene completeness, and sequencing technologies. Relative to species richness, genomic efforts have been biased toward four orders (Diptera, Hymenoptera, Collembola, and Phasmatodea), Coleoptera are underrepresented, and 11 orders still lack a publicly available genome assembly. The average insect genome assembly is 439.2 Mb in length with 87.5% of single-copy benchmarking genes intact. Most notable has been the impact of long-read sequencing; assemblies that incorporate long reads are ∼48× more contiguous than those that do not. We offer four recommendations as we collectively continue building insect genome resources: 1) seek better integration between independent research groups and consortia, 2) balance future sampling between filling taxonomic gaps and generating data for targeted questions, 3) take advantage of long-read sequencing technologies, and 4) expand and improve gene annotations

Long Reads Are Revolutionizing 20 Years of Insect Genome Sequencing

The first insect genome assembly (Drosophila melanogaster) was published two decades ago. Today, nuclear genome assemblies are available for a staggering 601 insect species representing 20 orders. In this study, we analyzed the most-contiguous assembly for each species and provide a "state-of-the-field" perspective, emphasizing taxonomic representation, assembly quality, gene completeness, and sequencing technologies. Relative to species richness, genomic efforts have been biased toward four orders (Diptera, Hymenoptera, Collembola, and Phasmatodea), Coleoptera are underrepresented, and 11 orders still lack a publicly available genome assembly. The average insect genome assembly is 439.2 Mb in length with 87.5% of single-copy benchmarking genes intact. Most notable has been the impact of long-read sequencing; assemblies that incorporate long reads are ∼48× more contiguous than those that do not. We offer four recommendations as we collectively continue building insect genome resources: 1) seek better integration between independent research groups and consortia, 2) balance future sampling between filling taxonomic gaps and generating data for targeted questions, 3) take advantage of long-read sequencing technologies, and 4) expand and improve gene annotations