Genes and calls reveal 5-fold greater diversity of Amazon frog species

Amazonian biodiversity has been studied for hundreds of years. Early explorers of Amazonian plants and animals included renowned naturalists of the stature of Alexander von Humboldt and A. R. Wallace. Despite this long history of exploration, new studies are resulting in the discovery of a large number of new species. The key of these discoveries lies in the use of advanced new tools for species detection.

The study, published in the open access journal ZooKeys, found up to 11 species among populations of what were previously considered two widespread treefrog species. Based on analyses of the genetic variation of dozens of Amazonian populations across six countries, the team lead by Marcel Caminer from the Museum of Zoology at Catholic University of Ecuador, found unequivocal evidence of the existence of a large amount of the so called “cryptic diversity“. The genetic results were corroborated with detailed analyses of male calls and body shape and color. The study formally describes four of the new species identified.

”These findings could not be possible without large-scale genetic sampling.” said Dr Santiago Ron, one of the authors of the study. ”The genetic data allows the discovery of species that have been hidden in museum shelves for decades. Genetic screening is opening a new age of scientific discovery in biodiversity studies in the Amazon region.”

Cryptic species” are two or more species mistakenly classified as a single one. Traditionally, taxonomists recognized species purely on morphological grounds and therefore failed to discriminate between species with similar appearance. The increasing use of DNA sequences for species recognition is demonstrating that current estimates vastly underestimate the true Amazonian species richness.

The discovery of cryptic diversity also has important implications for the conservation prospects of the species. ”What were considered two species with wide geographic distribution turned out to be eleven species with much smaller geographic ranges. This change implies that each species has a higher extinction probability.” said Dr. Ron. ”If our results are typical of Amazonian amphibians, a large scale reassessment of their conservation status and geographic distribution will be required.”

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Original Source:

Caminer MA, Ron SR (2014) Systematics of treefrogs of the Hypsiboas calcaratus and Hypsiboas fasciatus species complex (Anura, Hylidae) with the description of four new species. ZooKeys 370: 1. doi: 10.3897/zookeys.370.6291

Minimalistic raiding parties of a slave-hunting ant crack castles

A group of scientists from the University of Mainz and the Senckenberg Museum of Natural History Goerlitz, headed by Susanne Foitzik and Bernhard Seifert, recently described a new slave-making ant species from the eastern USA. They baptized the new ant Temnothorax pilagens – from pilere (Latin): to pluck, plunder or pillage. The paper was published in the open access journal ZooKeys.

In contrast to the famous slave-hunting Amazon Ants whose campaigns may include up to 3000 warriors, the new slave-maker is minimalistic in expense, but most effective in result. The length of a “Pillage Ant” is only two and a half millimeters and the range of action of these slave-hunters restricts to a few square meters of forest floor. Targets of their raiding parties are societies of two related ant species living within hollow nuts or acorns. These homes are castles in the true sense of the word – characterized by thick walls and a single entrance hole of only 1 millimeter in diameter, they cannot be entered by any larger enemy ant.

An average raiding party of the Pillage Ant contains four slave-hunters only, including the scout who had discovered the target. Due to their small size the raiders easily penetrate the slave species home. A complete success of raiding is achieved by a combination of two methods: chemical camouflage and artistic rapier fencing.

The observed behavior is surprising as invasion of alien ants in an ant nest often results in fierce, usually mortal, fighting. Here, however, in several observed raids of the Pillage Ant, the attacked ants did not defend and allowed the robbers to freely carry away broods and even adult ants to integrate them into the slave workforce. The attacked ants did not show aggression and defence because the recognition of the enemy was prevented by specific neutralizing chemical components on the cuticle of the slave-hunters.

The survival of slave ant nests is an ideal solution from the perspective of slave hunters as it provides the chance for further raids during the next years. In other observed raids chemical camouflage was less effective – perhaps because the attacked ant population was strongly imprinted to a more specific blend of surface chemicals. In fact, a defence reaction was more probable if the attacked colony contained a queen that causes a strong imprinting of chemical recognition cues.

If defending, the chance of a slave ant to win a fight with a  Pillage Ant is nearly zero. The attackers use their stinger in a sophisticated way, targeting it is precisely in the tiny spot where the slave ant’s neck is soft-skinned. This stinging causes immediate paralysis and quick death and may result in high rates of casualties ranging from 5% to 100% of the attacked nests’ population, whereas there are no victims among the attackers. If the Pillage Ants can conduct such successful raids with no or minimum own losses, there remains the question which factors regulate their population at a rather low level.

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Original Source:

Seifert B, Kleeberg I, Feldmeyer B, Pamminger T, Jongepier E, Foitzik S (2014) Temnothorax pilagens sp. n. – a new slave-making species of the tribe Formicoxenini from North America (Hymenoptera, Formicidae). ZooKeys 368: 65. doi: 10.3897/zookeys.368.6423

Museum bird DNA ‘ready for use’ in Naturalis Biodiversity Center

DNA barcoding is used as an effective tool for both the identification of known species and the discovery of new ones. The core idea of DNA barcoding is based on the fact that just a small portion of a single gene already can show that there is less variation between the individuals of one species than between those of several species.

Thus, when comparing two barcode sequences one can establish whether these belong to one single species (viz. when the amount of variation falls within the ‘normal’ range of the taxon under consideration and below a certain threshold level) or possibly to two species (when the amount exceeds this level).

A recent study in the open access journal ZooKeys sequenced 388 individuals of 147 bird species from The Netherlands. 95% of these species were represented by a unique barcode, but with six species of gulls and skuas having at least one shared barcode. This is best explained by these species representing recent radiations with ongoing hybridization. In contrast, one species, the Western Lesser Whitethroat showed deep divergences between individuals, suggesting that they possibly represent two distinct taxa, the Western and the Northeastern Lesser Whitethroat.

Our study adds to a growing body of DNA barcodes that have become available for birds, and shows that a DNA barcoding approach enables to identify known Dutch bird species with a very high resolution. In addition, some species were flagged up for further detailed taxonomic investigation, illustrating that even in ornithologically well-known areas such as the Netherlands, more is to be learned about the birds that are present.

“The barcoding approach is particularly useful in musea.”, comments Dr. Aliabadian, Ferdowsi University of Mashhad, Iran, “This illustrates the value of DNA tissue vouchers ‘ready for use’ from the bird collection of the Naturalis Biodiversity Center in Leiden.”

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Original source:

Aliabadian M, Beentjes KK, Roselaar CS, van Brandwijk H, Nijman V, Vonk R (2013) DNA barcoding of Dutch birds. In: Nagy ZT, Backeljau T, De Meyer M, Jordaens K (Eds) DNA barcoding: a practical tool for fundamental and applied biodiversity research. ZooKeys 365: 25–48. doi: 10.3897/zookeys.365.6287

DNA barcoding to monitor marine mammal genetic diversity

Marine mammals are flagship and charismatic species, very attractive for the general public. Nowadays, they are also considered as highly relevant sentinel of the marine realm. Their presence and their welfare in an area is thought to indicate the health of the place, whereas their disappearance, their displacement, or a decrease in their abundance or health could reflect negative environmental changes, whether of anthropogenic origin or not.

Monitoring marine mammal biodiversity is often difficult to perform. If some species can be easily observed, others are more difficult to detect, because for instance, of their scarcity or their discrete behavior. One of the solution suggested by scientists is based on the organization of stranding networks, listing and recording marine mammal strandings, which represent a cost-effective means to follow the marine mammal biodiversity.

Researchers from Océanopolis and from the Laboratory BioGeMME (Biologie et Génétique des Mammifères Marins dans leur Environnement) of the University of Brest, in collaboration with the Parc naturel marin d’Iroise and PELAGIS, have evaluated the usefulness of DNA barcoding in the monitoring of marine mammal biodiversity. They confirmed the species identifications performed by field correspondents, identified degraded carcasses or parts of carcasses, and examined intraspecific genetic variations for the harbour porpoise and the grey seal, undetectable by visual observation.

The conclusions of their study, published in a special issue of the open access journal Zookeys dedicated to DNA barcoding (DNA barcoding: a practical tool for fundamental and applied biodiversity research), are that the use of DNA barcoding in conjunction with a stranding network will clearly increase the accuracy of the monitoring of marine mammal biodiversity. Global climate change, as well as more localized environmental changes (some of which are caused by humans), has impacts on the marine realm. A routine use of DNA barcoding to monitor marine mammal biodiversity will clearly increase our capacity to detect such impacts, which is a necessary first step to take appropriate conservation measures.

In France, the French marine mammal stranding recording program has been created at the beginning of the 70s by the CRMM (Centre de Recherche sur les Mammifères Marins, La Rochelle, presently the Joint Service Unit PELAGIS, UMS 3462, University of La Rochelle- CNRS). The network comprises about 260 field correspondents, members of organizations or volunteers (Peltier et al. 2013, PloS One, e62180).

In Brittany (a region located at the northwest of France), the network is coordinated by Océanopolis in Brest. In this area, and all species included, an average of 150 animals strand each year, representing, in the last ten years, 14 species of cetaceans and five species of pinnipeds. These species include for instance, common and bottlenose dolphins, harbour porpoises, but also larger animals like minke whales and fin whales. Some rare stranding events include deep-diving or exotic species, such as arctic seals.

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Original source:

Alfonsi E, Méheust E, Fuchs S, Carpentier F-G, Quillivic Y, Viricel A, Hassani S, Jung J-L (2013) The use of DNA barcoding to monitor the marine mammal biodiversity along the French Atlantic coast. In: Nagy ZT, Backeljau T, De Meyer M, Jordaens K (Eds) DNA barcoding: a practical tool for fundamental and applied biodiversity research. ZooKeys 365: 5–24. doi:10.3897/zookeys.365.5873

Meloidogyne mali: A new invasive plant parasitic nematode in Europe

Following its recent synonymisation with Meloidogyne ulmi, a species known to parasitize elm trees in Europe, it has become clear that M. mali has been in the Netherlands for more than fifty years.

Evidences given by the authors suggest that M. mali was probably introduced during the breeding program on Elms against the Dutch Elm Disease (DED) during which large numbers of Elm rootstocks and seeds were imported from several different countries. The study was published in the open access journal ZooKeys.

M. ulmi
, the synonym of M. mali, was first described in Italy on the Elm tree species Ulmus Chenmoui. According to this current article, it has also been associated with several other plant species from a survey conducted in the Netherlands. It causes severe galling symptoms on its host, thereby interfering with their water and nutrient uptake from the soil.

On the current distribution of Meloidogyne mali in Europe, the authors speculate it could be found in ten other European countries namely Belgium, England, France, Ireland, Italy, Spain, Denmark, Germany, Slovakia and Romania. These were the European countries to which resistant Elm rootstocks were sent at the end of the breeding program.

The first description of M. mali was in Japan and it dates back to 1969, with the type host being apple. Many studies later on associated this nematode species with several plant species including Elms. In Europe, however it was for years only known to parasitize Elms. The trial field, ”Mierenbos”, in Wageningen used for growing the resistant Elm cultivars is completely infested with M. mali, with all the trees showing severe galling symptoms.

During this study, M. mali was tested on some more plant species which were found to be hosts to this nematode species. The authors compounded a list of about 44 different plant species currently recognized as host to M. mali. It is highly probable that this root-knot nematode has even a wider host range than what is known now, and possibly a wider distribution in Europe than where it is currently confined to be (Italy and the Netherlands).

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Original source:

Ahmed M, van de Vossenberg BTLH, Cornelisse C, Karssen G (2013) On the species status of the root-knot nematode Meloidogyne ulmi Palmisano & Ambrogioni, 2000 (Nematoda, Meloidogynidae). ZooKeys 362: 1–27. doi:10.3897/zookeys.362.6352

Tell me your barcode, and I will tell you what palm you are

Reliable and cost-effective species recognition is the dream of many scientists, and has important applications. While the use of morphological features is often uncertain, and can lead to misidentification, species identification based on the composition of short DNA sequences -the so-called “DNA barcodes“- has proven to be the safest way to reach this goal, both in animals and in many groups of plants.

Palms belonging to the genus Phoenix, including the economically-important date palm Phoenix dactylifera, i.e. the main fruit crop in North Africa and the Middle East, are amongst the groups of flowering plants characterized by difficulties in species discrimination based on their look. Moreover, given their high interfertility, they can easily hybridize whereas they come into contact, generating even more confusion for palm scientists.

To overcome such an issue, an international team of scientists examined a small region of chloroplast DNA, looking for a potential “barcode” for this group of plants. By screening over 130 palm individuals from 13 out of the 14 species of the genus Phoenix, they found enough variation in the composition of the DNA, to be able to identify correctly eight species out of 13, and more than 82% of the individuals. The study was published in the open access journal ZooKeys.

”It’s a very encouraging result.”, said Marco Ballardini, a biologist at that time research assistant at the Consiglio per la Ricerca e la Sperimentazione in Agricoltura (CRA-FSO) in Sanremo, Italy, and first author of this study. ”Finding the appropriate DNA barcode for Phoenix palms has several practical applications, ranging from the conservation of endemic and/or endangered species, like the Canary Island date palm, or the Cretan date palm, to the identification of hybrids having an ornamental value.”

The identification of palm individuals at the species level, as well as the detection of hybrids, can also be very helpful for preserving the genetic characteristics. Consequently, as in the case of date palm, the fruit quality of cultivated stocks, is one of the most interesting returns of this kind of study.

”To achieve a 100% success in identifying Phoenix palms, we have to analyze a few more regions of DNA, especially in the case of closely related species. Moreover, as the chloroplast DNA is inherited only through the maternal lineage, DNA of paternal origin should also be taken into consideration, in order to detect all possible hybrids.”, concluded Ballardini.

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Original source:

Ballardini M, Mercuri A, Littardi C, Abbas S, Couderc M, Ludeña B, Pintaud JC (2013) The chloroplast DNA locus psbZ-trnfM as a potential barcode marker in Phoenix L. (Arecaceae). In: Nagy ZT, Backeljau T, De Meyer M, Jordaens K (Eds) DNA barcoding: a practical tool for fundamental and applied biodiversity research. ZooKeys 365: 71–82. doi:10.3897/zookeys.365.5725

The value of museum collections for development of DNA barcode libraries

The ability to sequence the DNA of plants and animals has revolutionized many areas of biology, but the unstable character of DNA poses difficulties for sequencing specimens in museum collection over time. In an attempt to answer these issues, a recent study of 31 target spider species from the Naturalis Biodiversity Center in Leiden, discovers that both time and body size are significant factors in determining which specimens can produce DNA barcode sequences. The study was published in a special issue of the open access journal ZooKeys.

The specimens contained in the world’s natural history museums are the basis for most of what scientists know about biodiversity. Much like libraries, natural history museums are responsible for the long term preservation of their collections while circulating loans to active scientists. Museum curation techniques were developed over hundreds of years and optimized for anatomical preservation, and are often not ideal for preserving tissues for DNA sequencing.

DNA barcoding is an approach to the study of biodiversity that involves sequencing a standard region from the genome of an unidentified specimen and comparing it to a library of identified reference sequences representing many species. The success of this approach is in part dependent on the completeness of the library of reference sequences. When building such a reference library, specimens must either be freshly collected or taken from an existing collection.

The question addressed in this study is can we predict which specimens in a museum collection are likely to yield a successful DNA barcode sequence? If so, we can optimize our resources, wisely select museum specimens to sequence, and plan fresh collections to supplement. This study focused on Dutch spiders.

31 target species that have been frequently collected in the Netherlands over several decades and deposited in the Naturalis Biodiversity Center in Leiden were selected. For each target species, a series of increasingly older specimens was selected and brought to the lab for DNA sequencing. This was supplemented with freshly collected material representing nearly 150 Dutch spider species. The scientists recorded which specimens successfully produced DNA barcode sequences and which failed. They also experimented with DNA extraction techniques.

Typically, DNA extraction begins with the removal of muscle tissue; this is destructive extraction. An alternative approach is to soak the specimen in a solution that releases DNA from cells but does little or no damage to anatomy; this is nondestructive extraction. They found that failure rates for DNA barcode sequencing rise with time since collection, but body size is also a significant factor.

For freshly collected specimens overall, body size is not a predictor of sequencing success or failure. But larger species have a longer DNA barcoding shelf life than smaller species. Nondestructive extraction techniques can significantly improve the chances of obtaining a DNA barcode sequence. Considering only the commonly applied destructive extraction method, small spiders are useful for only a few years while those with a body length of around 3 mm or more have a good chance of yielding a barcode sequence for about 20 years after collection.

But using nondestructive extraction, even small spiders with a body length of 4 mm or less have a good chance of yielding a DNA barcode sequence for about 15 years after collection while spiders above this size can yield barcode sequences for a considerably longer time. The success of nondestructive extraction demonstrated here coupled with the need to preserve museum specimens for a variety of research purposes bodes well for museum collections are source material for DNA barcode libraries.

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Original Source:

Miller JA, Beentjes KK, van Helsdingen P, IJland S (2013) Which specimens from a museum collection will yield DNA barcodes? A time series study of spiders in alcohol. In: Nagy ZT, Backeljau T, De Meyer M, Jordaens K (Eds) DNA barcoding: a practical tool for fundamental and applied biodiversity research. ZooKeys 365: 245–261. doi: 10.3897/zookeys.365.5787

Describing biodiversity on tight budgets: 3 new Andean lizards discovered

Three beautiful new lizards from the Andes of Peru have been delimited and discovered using different lines of evidences by Peruvian and American biologists from San Marcos and Brigham Young universities respectively. The study was published in the open access journal Zookeys.

These lizards have been “hidden” and confused with other lizards of the same group because of their overall similar appearance. However this study, which includes molecular, ecological and more detailed morphological analyses, has identified them as new species. This research can be seen as an example of a midpoint between, on one extreme, species delimitation studies which rely on expensive molecular data and rarely include descriptions of new species, and at the other extreme, species descriptions that rely on morphology and rarely include more than elementary analyses.

The new study shows that with few resources, multiple different lines of evidence can be integrated to discover new species and provide a basis for more stable scientific names. Species with scientific names are crucial because they become “visible” to national and international governments and organizations devoted to biodiversity conservation. Species delimited but not formally described and without scientific names don’t exist in the real world, and this is an issue of pivotal importance in the Andean, Patagonian, and Neotropical regions of South America.

The new species are named for and dedicated to two different old Andean civilizations, Chavín and Wari, and an Inca ruler, Pachacutec. Liolaemus pachacutec was found above Písac, an Inca ruin built by Pachacutec. Liolaemus chavin was found in an area close to the center of the Chavín culture, where reptiles and other animals were represented in some remarkable artistic expressions. Liolaemus wari was found close to the center of Wari culture, in Ayacucho department, southeastern Peru.

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Original Source:

Aguilar C, Wood PL Jr, Cusi JC, Guzmán A, Huari F, Lundberg M, Mortensen E, Ramírez C, Robles D, Suárez J, Ticona A, Vargas VJ, Venegas PJ, Sites JW Jr (2013) Integrative taxonomy and preliminary assessment of species limits in the Liolaemus walkeri complex (Squamata, Liolaemidae) with descriptions of three new species from Peru. ZooKeys 364: 47. doi: 10.3897/zookeys.364.6109

 
Contacts:

César Aguilar
Email: caguilarp@gmail.com

Jack Sites
Email: jack_sites@byu.edu

2 of the oldest German journals in Zoology go for ‘platinum’ open access

Deutsche Entomologische Zeitschrift and Zoosystematics and Evolution join the family of Pensoft journals

Enough has been written and said about “platinum” open access as a step beyond the “green” and “gold” open access models. However, comparatively little has been seen of its practical implementation. On 1 January 2014, two of the oldest German journals in Zoology – Deutsche Entomologische Zeitschrift and Zoosystematics and Evolution – make a step right into the future by joining the journal publishing platform of Pensoft Publishers and adopting “platinum” open access.

For Pensoft, “platinum” open access means not just that the articles and all associated materials are free to download and that there are no author-side fees but even more so that novel approaches are used in the dissemination and reuse of published content. This publishing model includes:

  • Free to read, reuse, revise, remix, redistribute
  • Easy to discover and harvest by both humans and computers
  • Content automatically harvested by aggregators
  • Data and narrative integrated to the widest extent possible
  • Community peer-review and rapid publication
  • Easy and efficient communication with authors and reviewers
  • No author-side fees

Deutsche Entomologische Zeitschrift and Zoosystematics and Evolution are titles of the Museum für Naturkunde, Berlin. Deutsche Entomologische Zeitschrift, founded in 1857 as Berliner Entomologische Zeitschrift, is one of the oldest entomological journals worldwide, and the oldest one in Germany. It publishes original research papers in English on the systematics, taxonomy, phylogeny, comparative morphology, and biogeography of insects. Having long been indexed by Thomson Reuters’s Web of Science, now the journal will go on the route of innovation with Pensoft.

Zoosystematics and Evolution, formerly Mitteilungen aus dem Museum für Naturkunde in Berlin, Zoologische Reihe – is an international, peer-reviewed life science journal devoted to whole-organism biology, that also has a rich history behind itself (established in 1898). It publishes original research and review articles in the field of zoosystematics, evolution, morphology, development and biogeography at all taxonomic levels.

“Innovation is central to how Pensoft sees its role as a publisher. We want to make our articles as widely available and harvestable as possible both for the research community and the wider public. The attractive combination between historical legacy and tradition, advanced publishing technologies of Pensoft and free and open publishing models will ensure for these two authoritative journals a jump right into the future!” said the Managing Director of Pensoft Publishers, Prof. Lyubomir Penev.

“Making published data automatically harvestable by aggregators like the Global Biodiversity Information Facility or the Encyclopedia of Life is the only effective way how such databases can be increased and re-used to generate new knowledge. The Museum für Naturkunde is the first museum transferring its journals to the path of Pensoft Publishers and adapting them to such modern demands. I am excitedly looking forward to leading the Deutsche Entomologische Zeitschrift on this way.” said the new Editor-in-Chief of the Deutsche Entomologische Zeitschrift, Dominique Zimmermann from the Natural History Museum in Vienna.

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Additional Information:

Museum für Naturkunde – Leibniz Institute for Research on Evolution and Biodiversity is a research museum within the Leibniz Association. It is one of the most significant research institutions worldwide in biological and geo-scientific research on evolution and biodiversity. Established in 1810, its collections comprise more than 30 million items relating to zoology, palaeontology, geology and mineralogy, which are highly significant for science as well as for the history of science and at the centre of the research performed at the Museum.

Pensoft Publishers specialize in academic and professional book and journal publishing, mostly in the field of biodiversity science and natural history. In 2008, Pensoft launched its first open-access journals named ZooKeys and BioRisk, and since then then it has taken a leadership in introducing innovations in the field of open access. On 16th of September 2013, Pensoft launched the Biodiversity Data Journal (BDJ) and the associated Pensoft Writing Tool (PWT), as the first workflow ever to put together article authoring, community peer-review, publishing and dissemination within a single online collaborative platform.

 

Contacts:

Dominique Zimmermann
Editor-in-Chief of Deutsche Entomologische Zeitshrift
Natural History Museum Vienna
E-mail: dominique.zimmermann@nhm-wien.ac.at

Dr Matthias Glaubrecht
Editor-in-Chief of Zoological Systematics and Evolution
Natural History Museum Berlin
E-mail: Matthias.Glaubrecht@mfn-berlin.de

Dr Lyubomir Penev
Managing Director of Pensoft Publishers
E-mail: penev@pensoft.net

The economically valuable sweet-gum trees: Taxonomy and 9 new combinations

The sweet-gum family Altingiaceae is a small group of wind-pollinated trees that produce hard, woody fruits that contain numerous seeds. This widespread tree family has been puzzling botanists for a while, due to its complicated taxonomic structure, and the morphological similarities between the different genera which makes their separation and description a challenge. Best known for their biogeographic intercontinental disjunction between E Asia and E North America, recent molecular analysis have shown that Altingia and Semiliquidambar are nested within Liquidambar.

A new taxonomic synopsis, published in the open access journal PhytoKeys, formally transfers all Altingia and Semiliquidambar taxa to Liquidambar, which has nomenclatural priority and provides a new analysis including nine new combinations.

Traditionally classified into members with a predominantly temperate distribution (Liquidambar), those with a largely tropical to subtropical distribution (Altingia) are also presented in the new study, including the taxonomic enumeration and distribution of 15 recognized species based on studies of 1,500 specimens from 24 herbaria throughout the distributional range of the taxa.

Despite the difficulties in their taxonomy, sweet gum trees are in fact widely distributed and well known, due to their varied uses by people. They are valued for their high quality timber and they produce fragrant resin (styrax). Some species are also cultivated as ornamentals, while others are locally highly prized for the roots and bark used in traditional Chinese medicine. Some species are local endemics and Liquidambar chingii is listed as near-threatened by the IUCN.

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Original Source:

Ickert-Bond SM, Wen J (2013) A taxonomic synopsis of Altingiaceae with nine new combinations. PhytoKeys 31: 21. doi: 10.3897/phytokeys.31.6251