Diagnostic Morphological Characters Research Articles

A hairy giant among dwarves: Trichopelma grande, a distinct new species of tarantula from Cuba (Araneae: Theraphosidae)

ABSTRACT Large size, tropical climate, topographic complexity, and millions of years of isolation have turned the Greater Antillean islands into natural laboratories of evolution. Several groups have experienced explosive diversification across these islands, leading to highly diverse and endemic biotas and species experiencing unique adaptations. Here we describe a new tarantula species from the Viñales area, a biodiversity hotspot in western Cuba. Despite being a middle-sized species with large projected setae on the legs, reminiscent of those in arboreal tarantulas, phylogenomic analysis, diagnostic morphological features, and trap-door retreat building behaviour identifies it as the largest known representative of Trichopelma, and the only one with such hirsute legs. We provide mitochondrial barcoding data and a database of ultraconserved elements loci of the holotype of this species, which will help to include it in future phylogenetic studies of Theraphosidae. Given its limited distribution range, and its apparent absence in natural history collections despite its distinct appearance, this species might be scarce, positioning it as a potential conservation concern. http://www.zoobank.org/urn:lsid:zoobank.org:pub:2F9ED7A1-1345-4295-A876-6B617ABF7C75

The tooth-bearing skeletal elements of the Italian urodeles, a comparative tool for osteological identification

Urodele osteology is characterised by simplified skulls, loss of several bones and a specific sequence of cranial and limb ossification. The relatively few studies devoted to the comparative analysis of isolated urodele bones are mostly focused on the vertebrae and occipital complexes, and to a lesser extent humeri. The tooth-bearing skeletal elements (premaxillae, maxillae, dentaries, and vomers) are strongly neglected in this respect, despite being robust and as such sometimes found as fossils. Herein, we provide for the first time a comparative study of dentigerous bones, focusing on the Italian urodeles. Thirteen of the 19 species present in Italy, representing all genera except one, were analysed, for a total of 70 dry-prepared skeletons. The morphology of dentigerous skeletal elements of Italian urodeles is described and pictured, providing diagnostic characters and dichotomous keys for the identification at the genus level in most cases, and species level in some. The diagnostic morphological characters were included in a phylogenetic analysis, the results of which demonstrate that the tooth-bearing elements can have a phylogenetic value useful for assessing the relationships of living taxa

Anthomyza gilviventris in Palaearctic Region: integrative taxonomy, variability and habitat associations of North European population (Diptera: Anthomyzidae)

Anthomyza gilviventris Roháček & Barber, 2016, hitherto known only from the Nearctic Region, is recorded from the Palaearctic Region (NE Sweden) for the first time. Specimens from the Swedish population have been compared with those of A. gilviventris from Canada and the USA and those of A. tschirnhausi Roháček, 2009 from the Kamchatka Peninsula (Far East of Russia). Both morphological and molecular analyses (BI and RAxML, based on seven DNA markers: 12S, 16S, 28S, COI, COII, CytB, ITS2) confirmed that the Swedish specimens belong to A. gilviventris. Because no specimen of A. tschirnhausi has been available for molecular study, the most diagnostic morphological characters used for separation of this species from A. gilviventris have been re-evaluated with respect to Swedish specimens, and their variability discussed. However, these differences, although stable, are relatively small and, consequently, the possibility that they fall within the limits of a single variable species has not been entirely eliminated. New biological information (habitat and host-plant associations) on the Swedish population of A. gilviventris is presented.

Comparative plastome analysis of Arundinelleae (Poaceae, Panicoideae), with implications for phylogenetic relationships and plastome evolution

BackgroundArundinelleae is a small tribe within the Poaceae (grass family) possessing a widespread distribution that includes Asia, the Americas, and Africa. Several species of Arundinelleae are used as natural forage, feed, and raw materials for paper. The tribe is taxonomically cumbersome due to a paucity of clear diagnostic morphological characters. There has been scant genetic and genomic research conducted for this group, and as a result the phylogenetic relationships and species boundaries within Arundinelleae are poorly understood.ResultsWe compared and analyzed 11 plastomes of Arundinelleae, of which seven plastomes were newly sequenced. The plastomes range from 139,629 base pairs (bp) (Garnotia tenella) to 140,943 bp (Arundinella barbinodis), with a standard four-part structure. The average GC content was 38.39%, but varied in different regions of the plastome. In all, 110 genes were annotated, comprising 76 protein-coding genes, 30 tRNA genes, and four rRNA genes. Furthermore, 539 simple sequence repeats, 519 long repeats, and 10 hyper-variable regions were identified from the 11 plastomes of Arundinelleae. A phylogenetic reconstruction of Panicoideae based on 98 plastomes demonstrated the monophyly of Arundinella and Garnotia, but the circumscription of Arundinelleae remains unresolved.ConclusionComplete chloroplast genome sequences can improve phylogenetic resolution relative to single marker approaches, particularly within taxonomically challenging groups. All phylogenetic analyses strongly support the monophyly of Arundinella and Garnotia, respectively, but the monophylly of Arundinelleae was not well supported. The intergeneric phylogenetic relationships within Arundinelleae require clarification, indicating that more data is necessary to resolve generic boundaries and evaluate the monophyly of Arundinelleae. A comprehensive taxonomic revision for the tribe is necessary. In addition, the identified hyper-variable regions could function as molecular markers for clarifying phylogenetic relationships and potentially as barcoding markers for species identification in the future.

A redescription and new generic placement of the New Zealand marine-associated jumping spider Marpissa marina (Goyen, 1892) (Araneae: Salticidae: Euophryini)

ABSTRACT The jumping spider Marpissa marina (Goyen, 1892) is endemic to Aotearoa New Zealand. In this paper, we redescribe the species. Marpissa marina was placed in the Northern Hemisphere genus Marpissa due to its subjective similarity to spiders in that genus. The original description lacked modern morphological diagnoses and useful illustrations. The description was based largely on the female, and therefore lacked the taxonomically important characters seen in the male pedipalp. Previous literature has suggested M. marina may be related to the Australian genus, Maratus Karsch, 1878. We applied an integrative taxonomic approach based on diagnostic morphological characters and molecular analysis for a more detailed species description and assignment into an appropriate genus. Morphological features indicate that M. marina belongs to Maratus. This is supported by phylogenetic analyses using DNA sequences from the nuclear gene Actin 5C and the mitochondrial gene 16S ribosomal RNA. We transfer Marpissa marina into the genus Maratus, as Maratus marinus (Goyen, 1892), new combination.

DNA barcode reference library of the fish larvae and eggs of the South China Sea: taxonomic effectiveness and geographic structure

Fish early-stages constitute useful indicators of the states of marine ecosystems, as well as important fishery resources. Given the spectacular phenotypic changes during ontogeny, and the paucity of diagnostic morphological characters at the species level, the identification of fish early-stages is a challenging task. DNA barcoding, the use of the mitochondrial gene of the cytochrome c oxidase subunit I (COI) as an internal species tag, opened new perspectives for the identifications of both larval fish and fish eggs. However, the accuracy of the identifications assisted by DNA barcoding are dependent of the completeness of the DNA barcode reference libraries used to assigned unknown sequences to known species. Here, we built a DNA barcode reference library for 113 species of larval fish and 85 species of fish eggs involving the production of 741 newly generated DNA barcodes from South China Sea (63 localities). Together with 514 DNA barcodes mined from Genbank for 116 species from the South China Sea regions, a reference library including 1255 DNA barcodes for 308 species (248 locations) was assembled. The present study emphasizes the importance of integrating DNA barcoding to large scale inventories of early stages, as DNA-based species delimitation analyses delimited 305 molecular operational taxonomic units (MOTUs) and multiple cases of discordance with morphological identifications were detected. Cryptic diversity is detected with 14 species displaying two MOTUs and a total of 23 species were lumped into 11 MOTUs due to low interspecific divergence and/or mixed lineages.

Intraspecific variation of <em>Scorpaenopsis possi</em> Randall et Eschmeyer, 2002 (Pisces: Scorpaenidae)

The genus Scorpaenopsis has been intensively studied in recent decades. Sixteen species were known before the revision by Randall and Eschmeyer, who described 8 new species in 2002, and nowadays 29 species are known. Two specimens of Scorpaenopsis from the collection of the Zoological Institute of the Russian Academy of Sciences are intermediate in a number of parameters between Scorpaenopsis possi Randall et Eschmeyer 2002 and Scorpaenopsis eschmeyeri Randall et Greenfield 2004. The range of S. possi extends from the Indian Ocean coast of Africa and the Red Sea to the Marquesas Islands in the Pacific Ocean, the latitude of the range lies between the 30th latitudes of both hemispheres. The species S. eschmeyeri inhabits a much smaller range from the east coast of Australia to the island of Fiji. The aim of this work is to explain the appearance of S. possi, similar to S. eschmeyeri in some morphological diagnostic characters, almost 12,000 km apart from its range. The number of soft rays in the dorsal fin, which is unusual for the genus Scorpaenopsis, is described. Due to the lack of persistent distinguishing characters between S. possi and S. eschmeyeri, it is highly probable that both are conspecific; in that case S. eschmeyeri would be a junior synonym of S. possi.

A molecular approach to identify parrotfish (Sparisoma) species during early ontogeny.

Sparisoma species (parrotfish) comprise an important functional group contributing to coral-reef resilience. The morphological diagnostic characteristics for species identification are clearly described for adult forms but not for the early stages. Consequently, many taxonomical listings of Sparisoma larvae are restricted to the genus level. The aims of this study are to determine whether the morphological and molecular identification techniques are useful to assign the species taxonomic level to Sparisoma larvae occurring in the Gulf of Mexico and whether there is a set of diagnostic features that could be used to discriminate between species in larvae of different developmental stages. Morphological assignment of Sparisoma was performed based on morphological and meristic features for 30 larvae collected in the Gulf of Mexico from late August to mid-September 2015. To corroborate and complement the morphological assignments, molecular identification was carried out using DNA sequences from regions of two mitochondrial genes, mitochondrial cytochrome oxidase I (mtDNA COI) and mitochondrial 16S rRNA (mtDNA 16S rRNA). COI and 16S gene trees for Sparisoma and related fish taxa were constructed using sequences available in the NCBI (National Center for Biotechnology Information) GenBank and BOLD (Barcode of Life Data) databases. Two morphotypes were identified based on morphology, but no diagnostic characteristics for species discrimination were found. Molecular identification, in contrast, successfully discriminated four early development stages of Sparisoma atomarium, three stages of Sparisoma radians, and two stages of Sparisoma chrysopterum and Sparisoma aurofrenatum, therefore demonstrating the successful and necessary application of molecular taxonomic approaches for species-level identifications of Sparisoma larvae.

Redescription of the terrestrial isopod Armadillo mayeti Simon, 1885 and its comparison with Armadillo officinalis Duméril, 1816 from Tunisian populations

ABSTRACT The genus Armadillo is represented by 26 species, three of which are recorded from Tunisia: the Mediterranean A. officinalis Duméril, 1816, and the endemic A. mayeti Simon, 1885 and A. pseudomayeti Mattern, 1999. In this paper, the endemic species A. mayeti is redescribed, its morphological diagnostic characters are illustrated, and its stridulation apparatus structure and biochemical composition are highlighted for the first time using optical and scanning electronic microscopy. The stridulation apparatus, visible dorsally on propodus of pereopods 4 and 5, is structured in a row of chain scales. Regarding its chemical composition, the scale contains numerous elements with a high carbon content (C = 58.71%) followed by calcium (Ca = 19.63%), oxygen (O = 13.98), aluminium (Al = 4.81), copper (Cu = 1.02), iron (Fe = 0.63), potassium (K = 0.43), and silicon (Si = 0.20). Its distribution is extended and its status on the International Union for Conservation of Nature (IUCN) Red List is reported. Armadillo mayeti is then compared with the widespread Mediterranean A. officinalis for diagnostic purposes on the bases of morphological features and the stridulation apparatus structure focusing on the chemical composition. The taxonomic status of A. pseudomayeti is discussed.

DNA barcoding of southern African mammal species and construction of a reference library for forensic application.

Combating wildlife crimes in South Africa requires accurate identification of traded species and their products. Diagnostic morphological characteristics needed to identify species are often lost when specimens are processed and customs officials lack the expertise to identify species. As a potential solution, DNA barcoding can be used to identify morphologically indistinguishable specimens in forensic cases. However, barcoding is hindered by the reliance on comprehensive, validated DNA barcode reference databases, which are currently limited. To overcome this limitation, we constructed a barcode library of cytochrome c oxidase subunit 1 and cytochrome b sequences for threatened and protected mammals exploited in southern Africa. Additionally, we included closely related or morphologically similar species and assessed the database's ability to identify species accurately. Published southern African sequences were incorporated to estimate intraspecific and interspecific variation. Neighbor-joining trees successfully discriminated 94%-95% of the taxa. However, some widespread species exhibited high intraspecific distances (>2%), suggesting geographic sub-structuring or cryptic speciation. Lack of reliable published data prevented the unambiguous discrimination of certain species. This study highlights the efficacy of DNA barcoding in species identification, particularly for forensic applications. It also highlights the need for a taxonomic re-evaluation of certain widespread species and challenging genera.

Sychnotylenchus kulinichi sp. n. (Nematoda: Anguinoidea) from the Primorsky Krai of Russia with a tabular key to the genus Sychnotylenchus Rühm, 1956

Summary A new species of the genus Sychnotylenchus Rühm, 1956, S. kulinchi sp. n., inhabiting the bark beetle galleries of Pityogenes chalcographus in Pinus koraiensis was found in the Primorsky Krai of Russia. The new species differs morphologically from the closely related species S. yasinskii and S. mayor in having a shorter body length (626-1030 vs 1250-1280 and 1935-1455 μm, respectively) and from S. sparnugus in having a greater value of the ratio between spicule length and tail length (0.9-11 vs less than 0.7). Phylogenetic analysis of the partial 18S rRNA, D2-D3 expansion segments of the 28S rRNA, the ITS rRNA and COI gene sequences showed that the Russian S. kulinichi sp. n. population grouped with populations from Israel and an unspecified locality that were identified incorrectly as Ditylenchus forepolitor, which are considered conspecific with S. kulinichi sp. n. in this study. Phylogenetic analyses aiming to clarify the controversial taxonomic status of Neoditylenchus and Sychnotylenchus species indicated that, although the genus Sychnotylenchus was paraphyletic in some phylogenetic trees, testing of alternative topologies for the 18S rRNA, D2-D3 of 28S rRNA and ITS rRNA gene trees and datasets using the maximum likelihood method did not exclude the monophyly of this genus and did not justify the validity of the genus Neoditylenchus, which should be considered a junior synonym of Sychnotylenchus as proposed by Fortuner & Maggenti (1987). Molecular phylogeny showed close relationships of Sychnotylenchus with plant-parasitic nematodes from the genera Ficotylus, Orrina and Pterotylenchus, and also with the insect-associated genus Neomisticius. A polytomous key listing 24 morphological diagnostic characters for the identification of 33 species of Sychnotylenchus is provided, along with a table listing associated bark beetle species.

Unravelling the Brachidontes variabilis species complex (Bivalvia: Mytilidae) of the Indo-Pacific region

ABSTRACT Members of the radially ribbed mytilid genus Brachidontes are often dominant filter feeders on hard intertidal substrata in the Indo-Pacific region. However, there is still a great deal of uncertainty and confusion over their species identities, taxonomy and phylogenetic relationships. At least six species names (crebristriatus, karachiensis, mutabilis, pharaonis, ustulatus and variabilis) have at one time or another been synonymized with each other and applied to these common intertidal mussels. They occur widely across the Indian and Pacific Oceans from East Africa to Hawaii. Our results, based on sequences of five genes and anatomical features, suggest there are at least eight closely related sibling species in three monophyletic clades: (1) Brachidontes variabilis-form A in the western Indian Ocean; (2) Brachidontes pharaonis in the Red Sea; (3) Brachidontes variabilis-form B, found from India to East Asia; (4) Brachidontes mutabilis, restricted to southern Japan and China; (5) Brachidontes crebristriatus, in the Indian Ocean through to Southeast Asia and Hawaii; (6) Brachidontes ustulatus, an endemic species in western and southern Australia; (7) Brachidontes maritimus, so far known only from Hawaii; and (8) Brachidontes erosus, a southern Australian endemic. The latter two species form a small clade (Clade 1) that is sister to two larger Clades 2 and 3, each comprising three species. Clade 2 contains Brachidontes crebristriatus, Brachidontes mutabilis and Brachidontes ustulatus, whilst Clade 3 is made up of Brachidontes pharaonis, Brachidontes variabilis-form A and variabilis-form B. The shells of these eight species are not only variable but can greatly overlap in morphology. Many are also nearly impossible to distinguish without geographical information. However, the absence or presence of plicate organs in the mantle cavity, and the shape of guard papillae lining the inhalant aperture, may serve as important diagnostic morphological characters when used alongside geographical provenance.

Estudio palinológico de las Licofitas (Lycopodiopsida) de las Sierras Pampeanas centrales de Argentina

Background and aims: The Sierras Pampeanas Centrales of Argentina constitute the fourth centre of diversity of lycophytes and ferns in the Cono Sur, where representatives of four families of lycophytes live: Huperziaceae, Lycopodiaceae, Isoetaceae and Selaginellaceae. The aim of this work is to provide data of the morphology of their spores observed with optical and scanning electron microscopes, as a contribution to the taxonomy and conservation. M&M: The materials were analysed without prior chemical treatment and photographic records were obtained with both observation equipments. Results: The morphological diagnostic features of the spores are presented. The Huperziaceae and Lycopodiaceae are isosporous with trilete spores. Huperziaceae presents foveolated or fossulated spores, while Lycopodiaceae spores are reticulate, scabrate and baculate. In Isoetes hieronymi (heterosporate), the microspores are monolete with echinate ornamentation, while the megaspores are trilete, with reticulate ornamentation. The Selaginellaceae, all heterosporate, produce trilete microspores and megaspores, both with varied ornamentation. Conclusions: Spore characteristics provide relevant information for regional floristic studies as well as for taxonomic assignments, both of extant flora and fossil records. Also, for studies of the composition of soil spore banks as a tool for the conservation of local floristic biodiversity.

A new species of Neblinaphryne (Anura: Brachycephaloidea: Neblinaphrynidae) from Serra do Imeri, Amazonas state, Brazil

The highlands of the Guiana Shield (Pantepui) in northern South America harbor a unique fauna and flora. However, this diversity remains poorly documented, as many Pantepui massifs remain little explored or unexplored, mainly because their access is very challenging. Considering amphibians, 11 genera are endemic or sub-endemic to Pantepui, and one of them, Neblinaphryne, is monospecific and was recently described from the Neblina massif, at the border between Brazil and Venezuela. We recently undertook an expedition in the nearby, previously uninventoried Imeri massif and discovered a new species of this genus. We describe this new species herein as Neblinaphryne imeri sp. nov., combining molecular, external morphological, acoustic, osteological and myological data. The new species shares with the other Neblinaphryne species (N. mayeri) minuscule septomaxillae and pointed terminal phalanges, confirming the morphological diagnostic characters of the genus. Nevertheless, the new species can promptly be distinguished from N. mayeri by having the head wider than long, a distinct color pattern, and prominent tubercles on the eyelid and humeral region, as well as osteological and genetic differences. These two species are likely endemic to their respective massifs, providing a striking new example of speciation by isolation within Pantepui, which was possibly mediated by climate and elevation, as previously hypothesized for many other lineages endemic to this region.

Revision of the Neotropical Genus Solepyris Azevedo (Hymenoptera, Bethylidae).

The bethylid genus Solepyris Azevedo (Scleroderminae) is studied and revised. Five species are recognized, including three previously described ones: †S. electromexicanus Brazidec and Perrichot, S. montuosus Azevedo, and S. unicus Azevedo. Additionally, two new species are described and illustrated: S. maleku sp. nov. from Costa Rica and S. pataxo sp. nov. from Brazil. An emended diagnosis and a detailed discussion regarding the morphological diagnostic characters of this genus are also presented. A taxonomic key for all species is provided.

Investigation of the distribution patterns in moinids (Crustacea: Cladocera: Moinidae) forming ephippia with two resting eggs

During last two decades, morphological and genetic studies of the microcrustaceans from the family Moinidae Goulden, 1968 (Crustacea: Branchiopoda: Cladocera) were intensified. However, species diversity within this family remains underestimated. It refers to both subtropical and tropical areas of different continents that have traditionally been less studied compared with Central Europe and some other Palaearctic regions. In addition to this, only a restricted set of morphological characteristics is used for the discrimination of species within the genus Moina Baird, 1850. Most common moinid taxa are considered species complexes rather than single species, making any biogeographic reconstruction difficult. Here, we present an investigation of distribution patterns in moinids that form ephippia with two resting eggs based on both a reexamination of data from the literature and from specimens in museum and personal collections. Also, we redescribed morphology of poorly known taxa from the arid regions of the Old World—Moina belli Gurney, 1904 and M. kaszabi Forró, 1988 and clarify their diagnostic morphological characters and distributional ranges. We found that M. belli and M. kaszabi are morphologically very similar. In both species, setae 1 and 2 of thoracic limb I are armed bilaterally by fine, densely located short setulae. Reliable differences between M. belli and M. kaszabi concern armature of the valve posteroventral portion. Among the moinids with two resting eggs in the ephippium, M. belli and M. kaszabi are morphologically closer to M. macrocopa (Straus, 1820) and M. americana Goulden, 1968. All these species have: (1) a dorsal head pore; (2) fine long hairs on head, valve and preanal margin of postabdomen; (3) thoracic limb I of male with a long exopodite. In general, an investigated group of moinids includes both species with relatively compact and very broad distributional ranges. We concluded that maximum diversity of the moinids forming ephippia with two resting eggs is characteristic of southern portions of Palaearctic and Nearctic zoogeographical regions and approximately corresponds to subtropical climate zone. We need to underline that M. macrocopa and M. americana have a great invasive potential. Both species were anthropogenically introduced in South America and have been successfully naturalized there. Their distribution in tropical regions of the Old World and Australia requires new precise investigations. A group of moinids forming ephippia with two resting eggs can be considered as a convenient model for biogeographical reconstructions, including studies on changes in aquatic ecosystems due to global climate warming, eutrophication and the introduction of non-native species. There is no doubt that subsequent joint application of morphological and genetic data will shed more light on the global phylogeographic structure of the entire family Moinidae.

Revision of the Most Primitive Taxa of the Family Gyrodactylidae (van Beneden et Hesse, 1864) (Platyhelminthes, Monopisthocotyla) Based on ITS rDNA Phylogeny.

For the past 25 years, the ITS rDNA (ITS1-5.8S-ITS2) of Gyrodactylidae has been crucial for species identification, description, and phylogeny. This family includes 25 genera parasitizing marine and freshwater fish, initially distinguished by morphological differences in attachment and/or male copulatory organs. Gyrodactylus Nordmann, 1832, the most species-rich genus, has approximately 500 described species and an additional 25,000 species suspected. The genus is not monophyletic, and the functionally adaptive nature of morphological diagnostic characters complicates the delimitation of new genera. A phylogeny based on ITS rDNA was proposed to address these challenges, using only complete sequences of primitive taxa. Fifty-four sequences were aligned with the MUSCLE v5.1 algorithm, creating a 1590 ps long matrix. Maximum Likelihood (ML) and Bayesian Inference (BI) methods with the models TVM+F+G4 and SYM+G4 for ITS1-ITS2 and 5.8S, respectively, were inferred using IQ-TREE v2.3.5 and BEAST v2.7.6.0. The findings revealed eleven main lineages. Four of them are proposed for classification into new genera: Cichlidarus gen. nov., Iraqemembranatus gen. nov., Macracanthus gen. nov., and Rysavyius gen. nov. Elevating the subgenus G. (Gyrodactylus) to genus rank was supported. The presented phylogeny provides a foundation for developing a classification system within Gyrodactylidae that is both reasonable and comprehensive.

Overview of the Caucasian Perla Geoffroy, 1762 (Plecoptera: Perlidae) based on morphological and molecular data with description of two new species

Six species of Caucasian Perla are reviewed, and diagnostic morphological characteristics of all stages of development (where possible) are described, supplemented, and illustrated in detail with comparative light microscope and scanning electron microscopy images. The DNA barcoding of five species is presented. Two new morphologically and genetically distinct species, Perla schapsugica sp. nov. and Perla palatovi sp. nov., are described for both sexes and all life stages in the North Caucasus, Russia, Krasnodar Kray. Reinstatement of Perla persica Zwick, 1975, as a valid species distinct from P. caucasica Guérin-Méneville, 1843, is proposed. A new record of P. persica is reported for the Greater Caucasus, Russia, North-Ossetia-Alania for the first time. Morphologically, these two latter species can be separated in male adults by the shape of the hemitergal hook on terga X, an additional ventral brush on the penis of P. caucasica, wing length, and color.

Three New Truffle Species (Tuber, Tuberaceae, Pezizales, and Ascomycota) from Yunnan, China, and Multigen Phylogenetic Arrangement within the Melanosporum Group.

Based on a multi-locus phylogeny of a combined dataset of ITS, LSU, tef1-α, and rpb2 and comprehensive morphological analyses, we describe three new species from the Melanosporum group of genus Tuber and synonymize T. pseudobrumale and T. melanoexcavatum. Phylogenetically, the three newly described species, T. yunnanense, T. melanoumbilicatum and T. microexcavatum, differ significantly in genetic distance from any previously known species. Morphologically, T. yunnanense is distinctly different from its closest phylogenetically related species, T. longispinosum, due to its long shuttle-shape spores (average the ratio of spore length to spore width for all spores (Qm) = 1.74). Tuber melanoumbilicatum differs from the other species in having a cavity and long shuttle-shaped spores (Qm = 1.65). Although T. microexcavatum sampled ascomata have relatively low maturity, they can be distinguished from its closely related species T. pseudobrumale by the ascomata size, surface warts, and spore number per asci; additionally, phylogenetic analysis supports it as a new species. In addition, molecular analysis from 22 newly collected specimens and Genebank data indicate that T. pseudobrumale and T. melanoexcavatum are clustered into a single well-supported clade (Bootstrap (BS) = 100, posterior probabilities (PP) = 1.0); and morphological characteristics do not differ. Therefore, based on the above evidence and publication dates, we conclude that T. melanoexcavatum is a synonym of T. pseudobrumale. By taking into account current knowledge and combining the molecular, multigene phylogenetic clade arrangement and morphological data, we propose that the Melanosporum group should be divided into four subgroups. Diagnostic morphological features and an identification key of all known species in the Melanosporum group are also included. Finally, we also provide some additions to the knowledge of the characterization of T. pseudobrumale, T. variabilisporum, and T. pseudohimalayense included in subgroup 1 of the Melanosporum group.

Eyeing DNA barcoding for species identification of fish larvae.

Identification of fish larvae based on morphology is typically limited to higher taxonomic ranks (e.g., family or order), as larvae possess few morphological diagnostic characters for precise discrimination to species. When many samples are presented at any one time, the use of morphology to identify such specimens can be laborious and time-consuming. Using a reverse workflow for specimen sorting and identification leveraging high-throughput DNA sequencing, thousands of fish larvae can be DNA barcoded and sorted into molecular operational taxonomic units (mOTUs) in a single sequencing run with the nanopore sequencing technology (e.g., MinION). This process reduces the time and financial costs of morphology-based sorting and instead deploys experienced taxonomists for species taxonomic work where they are needed most. In this study, a total of 3022 fish larval specimens from plankton tows across four sites in Singapore were collected and sorted based on this workflow. Eye tissue from individual samples was used for DNA extraction and sequencing of cytochrome c oxidase subunit I. We generated a total of 2746 barcodes after quality filtering (90.9% barcoding success), identified 2067 DNA barcodes (75.3% identification success), and delimited 256 mOTUs (146 genera, 52 families). Our analyses identified specific challenges to species assignment, such as the potential misidentification of publicly available sequences used as reference barcodes. We highlighted how the conservative application and comparison of a local sequence database can help resolve identification conflicts. Overall, this proposed approach enables and expedites taxonomic identification of fish larvae, contributing to the enhancement of reference barcode databases and potentially better understanding of fish connectivity.